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 MethodsMethod comparisonEvaluated 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 evaluatedReference 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 assayReference 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 Kushnir2, Julie A Ray2, Peter Middleton2, Ravinder J Singh3, Alan L Rockwood1, and A Wayne Meikle1

1Department of Pathology, University of Utah, Salt Lake City, UT 841122ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108

3Department 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 VitD3 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.

Deming regression statistics

Winter 2008

(N=30)

Summer 2009

(N=97)

Winter 2009

(N=20)

2008-2009 (N=147)

slope 0.45 0.70 0.90 0.72intercept 4.8 4.6 -1.9 4.0

r 0.826 0.936 0.974 0.939slope 0.78 0.99 0.97 0.99

intercept -0.1 -1.2 -1.8 -1.3r 0.884 0.822 0.960 0.839

slope 0.93 0.93 0.93intercept -2.3 1.4 1.5

r 0.980 0.988 0.988slope 1.07

intercept -2.9r 0.963

Lab A, RIA

Lab A, CIA

Lab B, LC-MS/MS

Lab C, LC-MS/MS

References

IntroductionSlopeSlope 0.7180.718InterceptIntercept 3.9653.965RR 0.93940.9394

SlopeSlope 0.9880.988InterceptIntercept -1.344-1.344RR 0.83850.8385

SlopeSlope 0.9260.926InterceptIntercept 1.45441.4544RR 0.98820.9882

SlopeSlope 1.0741.074InterceptIntercept -2.9-2.9RR 0.96250.9625