Preanalytical Stability of 25(OH)–Vitamin D3 in Human Blood or Serum at Room Temperature: Solid as a Rock

In view of increasing interest in vitamin D, the New York Times recently suggested that it might become “the nutrient of the decade” (1). Vitamin D plays a major role in calcium and bone metabolism and is an essential (pre)hormone involved in cell maturation and proliferation (2). Vitamin D deficiency is linked to an increased risk of breast, colon, and prostate cancer as well as autoimmune diseases like juvenile diabetes (2). Analysis and method standardization of the key indicator of vitamin D homeostasis, 25(OH)–vitamin D3, has been studied (3)(4). Information about preanalytical stability of 25(OH)–vitamin D3 in human blood is hard to find, however, especially for “on the bench” conditions. One older study is incomplete and another focuses on freeze-thaw cycles (5)(6). Medical laboratory sample-handling guidelines currently require freezing the sample and protecting it from artificial light and repeated freeze-thaw cycles. On the other hand, food industries report very good stability of vitamin D in natural matrices like milk or fat. We tested the stability of 25(OH)–vitamin D3 in 8 different human blood samples under several sets of routine laboratory conditions. Because dietary supplementation of vitamin D in the Netherlands is confined to vitamin D3, we studied only 25(OH)–vitamin D3 stability. This restriction is a potential limitation of our study, but our aim was to investigate the natural form in human samples. Based on the close resemblance in structures of vitamin D2 and D3 and previous but limited experience with 25(OH)–vitamin D2 stability (5), we expected the stabilities for 25(OH)–vitamin D2 and D3 to be similar under the conditions of our study. We performed the study with leftover samples from 8 anonymous outpatients. Our study was approved by the hospital’s ethics committee. Samples were collected in BD plastic blood collection containers free of anticoagulant. The original 25(OH)–vitamin D3 concentrations ranged from 35–110 nmol/L. The baseline concentration values for our experiment (time = 0) were the means of quintuplicate analyses, and for subsequent measurements they were the means of triplicate analyses. After exposure to the specified conditions in the original BD container, serum sample aliquots in stoppered plastic tubes were frozen immediately at −20 °C until analysis. Storage conditions examined were extended common routine laboratory conditions: storage of serum at 6 °C and at room temperature (about 20 °C) in the dark and on the bench under usual artificial light conditions, and storage of unprocessed whole blood without anticoagulants at room temperature on the bench. Additionally, we tested the effect of multiple freeze-thaw cycles. 25(OH)–vitamin D3 measurements were performed on a Cobas E601 analyzer (Roche), using the vitamin D3 (25-OH) assay as recommended by the manufacturer. Samples from each storage experiment were analyzed in 1 run after being stored in the freezer at −20 °C. The interassay CVs for pooled serum analyses (frozen aliquots) were 6.9% at 25.5 nmol/L 25(OH)–vitamin D3 and 3.2% at 72.5 nmol/L. For the Roche Bone Control we found CVs of 4.8% at 49.8 nmol/L 25(OH)–vitamin D3 and 4.0% at 97.8 nmol/L. Exposure to 4 freeze-thawing cycles had no important effect on 25(OH)–vitamin D3 concentrations; the values for the mean of 8 samples increased 2.6% from the initial values. This small increase can be attributed to evaporation or freeze-drying processes, but we believe there is also a small positive effect due to increased turbidity of the samples after freeze-thawing cycles. A 4.0% decrease in the mean concentration was seen following storage at −20 °C for up to 2 months. Mean 25(OH)–vitamin D3 concentrations (n = 8) during storage under these common laboratory conditions are presented in Fig. 1 . A mean decrease of 2.3% was noted after 72 h storage of whole blood on the bench at room temperature, and a mean decrease of 3.4% after 24 h and 8.5% after 7 days storage of serum on the bench in daylight. Mean decreases of 4.5% after 3 days and 8.1% after 7 days storage of serum in the dark at room temperature were noted, whereas a mean decrease of 1.8% was observed after 7-day storage of serum in the refrigerator. Mean 25(OH)–vitamin D concentrations over time under various sample storage conditions expressed as percentage of the concentration at time (T) = 0. RT, room temperature. We found the concentrations of 25(OH)–vitamin D3 in its natural state bound to vitamin D–binding protein to be very stable at room temperature, even for unprocessed whole blood. For either processed or unprocessed samples, delays of several hours before analysis had negligible effects. Even “forgotten” samples or samples received in an unfrozen state appear to be suitable for analysis, because the decreases noted after 3 days under usual laboratory conditions at room temperature were less than the analytical interassay precision. There appears to be no need for serum to be frozen for transport, and whole blood might even be the specimen of choice for transport times up to 3 days. Storage conditions of serum at 4 °C for at least 7 days and up to 4 freeze-thawing cycles are permissible. 25(OH)–vitamin D3 concentrations seem to be “solid as a rock” at room temperature and under the common preanalytical conditions experienced in medical laboratories.