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

Abstract

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 D 3 , has been studied (3, 4 ). Information about preanalytical stability of 25(OH)-vitamin D 3 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 freezethaw 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 D 3 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 D 3 , we studied only 25(OH)-vitamin D 3 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 D 2 and D 3 and previous but limited experience with 25(OH)-vitamin D 2 stability (5 ), we expected the stabilities for 25(OH)-vitamin D 2 and D 3 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 D 3 concentrations ranged from 35-110 nmol/L. The baseline concentration values for our experiment (time ϭ 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 Ϫ20°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 RT, room temperature. 55:8 1584-1595 (2009) 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 Ϫ20°C for up to 2 months. Clinical ChemistryMean 25(OH)-vitamin D 3 concentrations (n ϭ 8) during storage under these common laboratory conditions are presented in Fig. 1. A mean decrease of 2.3% was noted after 72 h stora...

The Significance of Vitamin D Receptor Gene Polymorphisms for Susceptibility to Hepatocellular Carcinoma in Subjects Infected with Hepatitis C Virus

“…Catalog. No: 09002, Assay range: 0-200 ng/mL, Assay time < 2hours, Precision CV: < 10%, Storage: 2-8°C) [34]. Samples were measured in duplicates on a Tecan SLT Rainbow plate reader (Tecan, Männedorf, Switzerland).…”

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

“…According to this stability study, 25(OH) D levels were stable for 24 hours at 2-8°C, about 2 months at −20°C, and 3 months at −40°C. Other studies have indicated that vitamin D was a stable analyte under common storage conditions [7][8][9][10][11]. Some authors investigated the freeze-thaw process and whether the samples were affected by sunlight or artificial light.…”

“…Some authors investigated the freeze-thaw process and whether the samples were affected by sunlight or artificial light. Wielders et al [7] examined blood samples from 8 individuals at 6°C, at room temperature protected from light, and at room temperature exposed to artificial light. They also analyzed the effect of 4 freeze-thaw cycles.…”

“…In that study, it was reported that serum vitamin D levels did not change at 4°C for at least 7 days and up to 4 freeze-thaw cycles. The authors reported the stability of vitamin D at room temperature as "solid as a rock" [7]. In a similar study, vitamin D levels were measured with a binding method and 11 freeze-thaw processes, and no significant change was detected in the measurement results [8].…”

Low Vitamin D Serum Levels May Be a Modifiable Risk Factor for Obesity and Cognitive Impairment in Middle-Age Egyptian Women

“…-According to Li et al (2004) [22], insulin resistance was expressed by modified homeostasis model assessment-insulin resistance (M.HOMA-IR) =1.5+ FBG (mg/dl) ×fasting C-peptide (ng/ ml) / 2800, in which insulin was replaced by C-peptide so as to be applied on diabetic patients using exogenous insulin. -Serum 25 hydroxy vitamin D (25 (OH) D) was assessed by Vitamin D direct ELISA kit (EIA-4696) DRG ® International, Inc. USA [23]. -Serum parathyroid hormone (PTH) was measured by human PTH ELISA kit (hPTH-EASIA) KAP1481 DIAsource ImmunoAssays S.A. -Rue de l'Industrie, 8 -B-1400 Nivelle -Belgium [24].…”