Effect of Anticoagulants and Storage Temperature on the Stability of Receptor Activator for Nuclear Factor-κB Ligand and Osteoprotegerin in Plasma and Serum

Abstract

The discovery of receptor activator for nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) as the fundamental factors controlling osteoclast formation and activation has advanced the understanding of the processes involved in osteoclastogenesis and bone remodeling (1)(2)(3). RANKL is important for osteoclast survival, differentiation, maturation, and activation, whereas OPG opposes these actions. Expression of RANKL and OPG is altered in many bone remodeling disorders, suggesting that determination of the role of these proteins in bone diseases is of value in understanding their etiology (4). Accurate quantification of sRANKL and OPG concentrations in serum samples is paramount in research involving metabolic bone disease. The availability of ELISAs for both sRANKL and OPG has led to investigation of the concentrations of these proteins in human samples collected from patients with several disorders and has allowed monitoring of the effect of treatment in bone-related diseases (4)(5)(6)(7)(8)(9). Previous reports of OPG concentrations in postmenopausal women with osteoporosis have produced discordant results in relation to bone turnover (5)(8). Other reports using the sRANKL:OPG ratio to estimate the extent of Paget disease of bone and the effect of bisphosphonate treatment have also shown inconclusive and inconsistent results (9)(10)(11). These data suggest a possible variability in sRANKL and OPG measurements in human serum or plasma. In this study we aimed to clarify whether this variability is attributable to genuine differences among the groups of patients studied or whether it reflects inaccuracies resulting from the sampling process. We investigated several factors that may influence the concentrations of sRANKL and OPG in human blood samples by (a) collecting blood into different anticoagulants; (b) varying the time between blood collection and plasma/serum separation; (c) altering the length of storage of …