Pre-analytical stability of 25 hydroxy vitamin D in human serum

21 Berna Bozkurt, Stability of 25-hydroxy vitamin D / doi: 10.14744/ijmb.2017. 08208 The aim of this study was to evaluate the effect of different time intervals and storage temperature conditions on samples to simulate circumstances in which they would not be run on the day of collection and be transported to an external laboratory. Materials and Methods The study was performed in the Sakarya Training and Research Hospital Biochemistry Department, in Sakarya, Turkey, between July and December 2014. A total of 153 individuals (125 female, 28 male) were included in the study. Subjects were healthy adults, with no known diagnosis of cancer, diabetes mellitus, cardiovascular disease, or osteoporosis. Information about the study was provided to all participants, and all completed an informed consent form. The study was conducted according to the principles of the Declaration of Helsinki and was approved by the Sakarya University Faculty of Medicine ethics committee (no.: 16.03.2015-3308). Venous blood was processed into 5-mL serum separator tubes with clot activator (BD Vacutainer; Becton Dickinson and Co., Franklin Lakes, NJ, USA). All collection tubes were centrifuged at 4000 xg for 5 minutes in a refrigerated centrifuge after the serum samples had been at room temperature for 30 minutes to allow proper clot formation. After centrifugation, all of the serum samples were aliquoted into 4 vials. One aliquot was analyzed immediately after collection (0hour sample) and accepted as the reference for the comparison with the other aliquots. The time intervals and storage conditions for the aliquots were Group 1: 1 vial for 0-hour time measurement, Group 2: 24 hours at 2-8°C, Group 3: about 2 months at -20°C, and Group 4: 3 months at -40°C. The level of 25(OH)D was determined using an automated acridinium ester magnetic particle chemiluminescence method on an IDS-ISYS analyzer and manufacturer reagents (Immunodiagnostic Systems, The Boldons, UK). Prior to determination of vitamin D concentration for each study period, the device was calibrated with the original calibrator and verified with internal quality control serum (IDS-ISYS 25-Hidroxy Vitamin D Control Set; Immunodiagnostic Systems, The Boldons, UK). All samples were run with reagents of the same lot number. Internal quality control samples were included in the assay run, and intraassay coefficients of variation (CV) values were obtained from the measurements. The intra-assay CV was 7.66%, 5.81%, and 4.92%, with target values of 19.5 ng/mL, 33.4 ng/mL, and 66.0 ng/mL (IDS-ISYS 25-Hydroxy Vitamin D Control L, M, and H; Immunodiagnostic Systems, The Boldons, UK ). The specimens were exposed to standard lighting conditions in the laboratory and there were no changes in the lighting conditions throughout the study. As soon as blood was drawn from the patients, it was transferred without being covered with laboratory foil and was exposed to direct daylight. Statistical Analysis Statistical data were processed using PASW Statistics for Windows, Version 18 (SPSS Inc., Chicago, IL, USA). The conformity of variables to normal distribution was tested with the Kolmogorov-Smirnov test. The descriptive statistics of continuous variables were expressed as mean±SD or median (minmax). In the tube group analysis, the presence of a statistically significant difference between the groups was examined with a paired t-test for parametric and the Wilcoxon signed rank test for nonparametric continuous variables. P values <0.05 were considered statistically significant.