Stability of Specimens for N-mid Osteocalcin and intact Osteocalcin Assays and Correlation of Results between the Two Assays

Abstract

Background : Serum osteocalcin has been known to be the marker of bone metabolism. NMIDTM Osteocalcin One Step ELISA (BioTech A/S, Denmark, for N-mid OC) measures intact osteocalcin (1-49 a.a.) including N-terminal midfragments (1-43 a.a.) whereas NovoCalcin (Metra Biosystems, USA, for intact OC) measures intact osteocalcin. We evaluated the stability of specimens and the correlation both assays. Method : We used the sera of 189 women over 40 years old for the correlation of N-mid OC and intact OC. The following specimens were prepared to evaluate the stability of specimens: whole blood and serum specimens were allowed to stand at ambient temperature for 0, 3 and 6 hours. Pooled sera from 3 apparently healthy individuals were stored at 41C, -201C and -701C and were then subjected to be assayed in 0, 1, 2, 4 weeks after the storage. The assays were done in triplicate in a single analytical batch. Results : The means±S.D. (ng/ml) of N-mid OC and intact OC were 9.79±5.76 and 6.43± 3.71, respectively. The correlation of both assays were as follows, r=0.79 (P<0.005), and [intact OC] = 0.51 x [N-mid OC] + 1.45. The storage of whole blood samples at room temperature showed gradual decrease of results from both assays, i.e. 91 % and 89% of baseline value 3 and 6 hours after for N-mid OC and 79% and 75% for intact OC. However, the serum samples were stable at room temperature for the same duration; 107% and 98% for N-mid OC and 94% and 103% for intact OC, respectively. The results of 1, 2 and 4 weeks after storage of serum specimens at 41C showed a significant decrease; 76%, 62% and 54% for N-mid OC and 21%, 9% and 0% for intact OC; those at -20´C showed 110%, 108% and 77% for N-mid OC and 111%, 85% and 105% for intact OC; and those at -701C showed 108%, 107% and 92% for N-mid OC and 108%, 95% and 110% for intact OC. Conclusions : The results of serum N-mid OC correlate well with that of intact OC. The serum specimens were stable at room temperature for 6 hours, but whole blood samples were unstable. Therefore, serum should be separated without delay after phlebotomy. Serum should be frozen at - 701C for storage longer than 1 week.