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Technical Briefs |
1 Division of Endocrinology, Department of Medicine, and 2 Department of Epidemiology and Biostatistics, University of California, San Francisco, 74 New Montgomery St., Suite 600, San Francisco, CA 94105
aauthor for correspondence: fax 415-597-9213, e-mail dantoniucci{at}psg.ucsf.edu
Vitamin D deficiency is a common disorder believed to affect as many as 57% of hospitalized patients (1) and 9–50% of outpatients, depending on the characteristics of the study population (2)(3)(4)(5)(6). Vitamin D deficiency can cause secondary hyperparathyroidism and, if severe, osteomalacia. Both of these conditions are associated with loss of bone density, and both are reversible by repletion of vitamin D (7)(8)(9)(10). Recently, the association of vitamin D deficiency with an increased risk for a broader range of diseases, such as prostate cancer (11)(12) and colon cancer(13), has come to attention. Dietary vitamin D supplementation has even been implicated in decreasing the risk of developing type 1 diabetes mellitus (14), and ultraviolet irradiation has been shown to decrease systolic blood pressure in patients with mild untreated hypertension (15). Determining whether vitamin D deficiency is present usually involves assessing the stores of vitamin D by measuring serum 25-hydroxyvitamin D (25OHD) concentrations, in combination with an assessment of parathyroid hormone concentrations. Currently, the preferred clinical 25OHD assay is a two-step procedure that involves a rapid extraction step of 25OHD from serum or plasma, followed by an equilibrium RIA procedure. The RIA is based on an antibody with specificity toward 25OHD. Specimens are obtained and immediately frozen at –20 °C or lower, then thawed just before the assay is performed. Vitamin D analytes, however, appear to be quite stable even at other temperatures, as was demonstrated by the International Quality Assessment Scheme for Vitamin D metabolites (DEQAS), a multinational study run out of the UK. Studies in the laboratory of the DEQAS organizer have shown no significant change in results for vitamin D metabolites during storage for up to 2 weeks at 30 °C (16). Consequently, DEQAS samples of vitamin D metabolites are sent to participants at ambient temperature, by first class mail, and typically arrive within 2 weeks of dispatch without any evidence of deterioration of the vitamin D metabolites.
Assaying serum for 25OHD is relatively expensive and is therefore often forfeited in large clinical trials of osteoporosis drugs. Undetected vitamin D deficiency may increase a patient’s risk of hypocalcemia when treated with bisphosphonate therapy (17). Many prospective studies have stored serum from participants and therefore offer the opportunity to further study the relationship between vitamin D deficiency and diseases such as colon, breast, and prostate cancer and diabetes. However, the serum samples that are stored as part of these large epidemiologic studies are often limited and desired by multiple investigators, and not uncommonly have been thawed and refrozen at least once. There are no published data about the reproducibility of vitamin D assays in serum samples that have undergone multiple freeze-thaw cycles. Experts often informally state that the assay should be reliable when performed in samples that have undergone up to three freeze-thaw cycles [Drs. M.F. Holick (Boston University Medical Center, Boston, MA) and R. R. Recker (Creighton University, Omaha, NE), personal communications]. The instruction manual that accompanies the DiaSorin 25OHD assay reports that no significant changes in values were observed for samples subjected to three freeze-thaw cycles (18). This information is based on internal data from DiaSorin, which studied serum 25OHD concentrations in five individuals. The concentrations were measured at baseline, then after three freeze-thaw cycles, and the correlation between these two measurements was 0.61. We feel that this correlation coefficient is too low to comfortably use samples that have previously been thawed. Furthermore, the data do not provide information on the correlation between the baseline value and each subsequent value. For these reasons, we sought to determine the correlation between baseline serum 25OHD concentrations and those obtained after the same samples underwent each of three freeze-thaw cycles.
We recruited 20 healthy adults. All participants gave informed consent before participation in the study, which was approved by the University of California, San Francisco (UCSF), Institutional Review Board. Individuals were excluded if they were on medications or had medical conditions known to alter vitamin D metabolism, such as malabsorption conditions, renal or hepatic disease, therapy with vitamin D exceeding the d ietary recommended intake (19), or therapy with antiepileptic medications. A single serum sample was collected from each participant and immediately aliquoted into four vials. All four vials were frozen at –70 °C. We define each freeze-thaw cycle (FT) as the vial being frozen and thawed once. The four collected vials were exposed to one, two, three, or four freeze-thaw cycles and were maintained fully frozen for 3 days between thaws. This meant that one vial was never thawed until the day of the 25OHD assay performance (FT1), one vial was thawed once (FT2), one vial was thawed twice (FT3), and one was thawed three times and refrozen (FT4) before the day of the 25OHD analysis. Specimens were analyzed by the UCSF clinical laboratory by use of the commercially available DiaSorin RIA with antibodies specific to 25OHD.
Correlation coefficients for the 25OHD result were calculated between the FT1 and each subsequent freeze-thaw cycle, with a P <0.05 considered as significant. All analyses were performed by use of Stata 8.0 (Stata Corporation).
Among the participants, 13 were women and 7 were men; the mean age was 39.8 years (range, 23–60 years). The median (interquartile range) 25OHD concentration in the baseline specimen was 57.4 (39.9–73.6) nmol/L (Table 1
). The correlation between the baseline 25OHD measurement and the measurement obtained after each successive freeze-thaw cycle was 0.99 (Fig. 1
).
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Our data show that 25OHD results obtained for specimens that have been thawed and refrozen up to four times are reliable. The data also suggest that investigators can use stored specimens from epidemiologic studies to measure 25OHD even if the samples have been previously thawed for other purposes.
Acknowledgments
These studies were carried out in part in the General Clinical Research Center, Moffitt Hospital, University of California, San Francisco, with funds provided by the National Center for Research Resources, 5 M01 RR-00079, US Public Health Service. This study was also supported by NIH Grant T-32 DK07418.
References
The following articles in journals at HighWire Press have cited this article:
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  J. P. M. Wielders and F. A. Wijnberg Preanalytical Stability of 25(OH)-Vitamin D3 in Human Blood or Serum at Room Temperature: Solid as a Rock Clin. Chem., August 1, 2009; 55(8): 1584 - 1585. [Full Text] [PDF]
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S. Mark, K. Gray-Donald, E. E. Delvin, J. O'Loughlin, G. Paradis, E. Levy, and M. Lambert Low Vitamin D Status in a Representative Sample of Youth From Quebec, Canada Clin. Chem., August 1, 2008; 54(8): 1283 - 1289. [Abstract] [Full Text] [PDF]
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 M. L Neuhouser, B. Sorensen, B. W Hollis, A. Ambs, C. M Ulrich, A. McTiernan, L. Bernstein, S. Wayne, F. Gilliland, K. Baumgartner, et al. Vitamin D insufficiency in a multiethnic cohort of breast cancer survivors Am. J. Clinical Nutrition, July 1, 2008; 88(1): 133 - 139. [Abstract] [Full Text] [PDF]
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 E. Hypponen, S. Turner, P. Cumberland, C. Power, and I. Gibb Serum 25-Hydroxyvitamin D Measurement in a Large Population Survey with Statistical Harmonization of Assay Variation to an International Standard J. Clin. Endocrinol. Metab., December 1, 2007; 92(12): 4615 - 4622. [Abstract] [Full Text] [PDF]
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 P. Tuohimaa, L. Tenkanen, H. Syvala, S. Lumme, T. Hakulinen, J. Dillner, and M. Hakama Interaction of Factors Related to the Metabolic Syndrome and Vitamin D on Risk of Prostate Cancer Cancer Epidemiol. Biomarkers Prev., February 1, 2007; 16(2): 302 - 307. [Abstract] [Full Text] [PDF]
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