1. Vitamin D (Vit D) is produced endogenously by exposure of skin to sunlight, and is absorbed from foods containing or supplemented with Vit D. Vit D is first metabolized in the liver to produce 25-OH Vit D. It is then metabolized into its biologically active form by the kidneys, providing 1,25-OH Vit D, a hormone that regulates calcium and phosphate metabolism. Deficiency of Vit D may result in impaired formation of bone, because of mineralizing defect, producing rickets in children and osteomalacia in adults. Research has also suggested that Vit D is protective against osteoporosis, hypertension, cancer, and several autoimmune diseases. Populations at risk for Vit D deficiency Elderly Obese individuals Exclusively breastfed infants Individuals with: limited sun exposure fat malabsorption syndromes Inflammatory bowel disease Study objective To compare commercially available 25-OH VitD assay methods: RIA, CIA, and LC-MS/MS assays. Seasonal variation was considered in the comparison. Comparison of methods for measurement of 25-hydroxy-vitamin D Materials and Methods Method comparison Evaluated using National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 972 and 147 specimens that were obtained following completion of clinical testing. Specimens were collected in the winter 2008 (N=30) and winter 2009 (N=20) and the summer (2009, N=97). Specimens chosen contained 25-OH VitD concentrations spanning the range of <5 to 149 ng/mL as measured by the LIAISON® 25 OH Vitamin D TOTAL Assay by DiaSorin that uses chemiluminescent immunoassay (CIA) technology. In addition, adequate specimen volume was required to make five-500 μL aliquots for testing by each laboratory. Laboratory testing methods evaluated Reference Laboratory A used the following 3 methods: (1) DiaSorin 25-OH-D assay by radioimmunoassay, (2) DiaSorin LIAISON® 25 OH Vitamin D TOTAL Assay by CIA (3) LC-MS/MS assay** Reference Laboratory B used a LC-MS/MS assay Reference Laboratory C used a LC-MS/MS assay ** All LC-MS/MS methods measured D2 and D3 separately, the total was then calculated. Lab A, LC-MS/MS method is the comparison method. JoDell E Whittington *,1, Mark M Kushnir 2, Julie A Ray 2, Peter Middleton 2, Ravinder J Singh 3, Alan L Rockwood 1, and A Wayne Meikle 1 1 Department of Pathology, University of Utah, Salt Lake City, UT 84112 2 ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108 3 Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905 Results All results were compared to the LC-MS/MS 25-OH VitD assay results of laboratory A (Table 1). Deming regression analysis gave a range of slopes (0.45 to 1.07) and correlation coefficients (r, 0.822 to 0.988). The CIA method used in Lab A showed marked improvement from winter of 2008 to summer & winter of 2009. Simultaneously, a marked improvement in the overall performance of RIA was observed over the same time period. Improvements may be due to differences in the sample sets being tested or due to small changes made by the manufacturer of the commercially available assays. In addition to patient samples, NIST standard reference material (972) was tested by CIA and LC-MS/MS and measured within 10% of the expected concentration of 25-OH VitD. Influence on 25-OH VitD 3 production Latitude Season Aging Sunscreen use Skin pigmentation Conclusions Overall, a negative bias is observed when RIA is compared to CIA and LC-MS/MS methods. The CIA method is in good agreement with LC-MS/MS 25-OH VitD assays. Lab improvements The introduction of NIST material, specifically level 1 made using undiluted human serum, provides a great measure of accuracy in assessment of the 25-OH VitD assays. NIST standard reference material (972) was tested by CIA and LC- MS/MS and measured within 10% of the expected concentration of 25-OH VitD TABLE 1 : Deming Regression statistics of 25-OH Vit D assay method comparisons with LC-MS/MS method, Lab A Burtis CA, Ashwood ER, Tietz NW. Tietz textbook of clinical chemistry. 4th ed. Philadelphia: W.B. Saunders, 2006, 1920-1926. References Introduction Slope0.718 Intercept3.965 R0.9394 Slope0.988 Intercept-1.344 R0.8385 Slope0.926 Intercept1.4544 R0.9882 Slope1.074 Intercept-2.9 R0.9625