25OH Vitamin D Total Assay - University of California, San Francisco

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I. PURPOSE UNIVERSITY OF CALIFORNIA SAN FRANCISCO, CLINICAL LABORATORIES The LIAISON 25 OH Vitamin D TOTAL Assay uses chemiluminescent immunoassay (CLIA) technology for the quantitative determination of 25-hydroxyvitiamin D and other hydroxylated vitamin D metabolites in human serum to be used in the assessment of vitamin D sufficiency. Assay results should be used in conjunction with other clinical or laboratory data to assist the clinician in making individual patient management decisions in an adult population. SUMMARY AND EXPLANATION OF TEST The role of vitamin D in bone and mineral metabolism was recognized from its first identification as a factor that could cure rickets. However, vitamin D is now recognized as a prohormone, which has multiple roles in maintaining optimal health. Vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol) are the most abundant forms of vitamin D in the body. Vitamin D is synthesized in the skin from 7-dehydrocholesterol in response to sunlight. The best nutritional sources of D3 are oily fish, primarily salmon and mackerel. Vitamin D2‘s nutritional sources are some vegetables, yeast and fungi. The vegetarian diet is abundant in vitamin D2. Vitamin D (D3, D2 and metabolites) is converted to 25-hydroxyvitamin D in the liver. The measurement of 25-OH vitamin D concentration in the serum or plasma is the best indicator of vitamin D nutritional status. The optimal level of 25-OH vitamin D is subject to some debate, but >32ng/mL (>80nmol/L) is accepted as sufficient for bone health. Vitamin D toxicity is a recognized problem, but a rare occurrence. Instead, a recent growing public health problem is vitamin D insufficiency. Several research studies have identified widespread vitamin D insufficiency in apparent healthy populations world-wide. Vitamin D deficiency is commonly treated with vitamin D2 or D3 medications in doses ranging from 50,000 IU/month to 50,000 IU/week. Fortified foods and nutrition supplements may contain either form. To ensure accurate assessment of the total vitamin D all forms, including D3, D2 and metabolites, must be measured. Recent studies have identified an inactive 3-epimer of 25-OH vitamin D, which may be present in the serum of infants under the age of one year. Thus it is important that an assay avoid measuring the inactive 3-epi form and only measure the active D3 and D2 forms equally. BIOLOGICAL PRINCIPLES OF THE PROCEDURE The LIAISON 25 OH Vitamin D assay is a direct competitive chemiluminescence immunoassay (CLIA) for quantitative determination of total 25 OH vitamin D in serum. During the first incubation, 25 OH vitamin D is dissociated from its binding protein and binds to the specific antibody on the solid phase. After 10 minutes, the tracer (vitamin D linked to an isoluminol derivative) is added. After a second 10 minute incubation, the unbound material is removed with a wash cycle. Subsequently, the starter reagents are added to initiate a flash chemiluminescent reaction. The light signal is measured by a photomultiplier as relative light units (RLU) and is inversely proportional to the concentration of 25 OH vitamin D present in calibrators, controls or samples. II. POLICY/SCOPE This procedure is intended for use by licensed CLS staff in the China Basin Chemistry section. III. TEST AVAILABILITY Test performed Monday, Wednesday and Fridays. Results will be available by 16:00. eterr:Library:Mail Downloads:25OHVitDTotal.doc Page 2 of 15
IV. SPECIMEN REQUIRMENTS SPECIMEN TYPES UNIVERSITY OF CALIFORNIA SAN FRANCISCO, CLINICAL LABORATORIES 1. Human serum or SST tubes. 2. Fasting samples are recommended, but not required. 3. Grossly hemolyzed or lipemic samples as well as samples containing particulate matter or exhibiting obvious microbial contamination should not be tested. 4. The Diasorin LIAISON does not provide the capability to verify specimen type. It is the responsibility of the operator to verify that the correct specimen type is used in the 25OH Vitamin D TOTAL assay. PREPARATION FOR ANALYSIS STORAGE STABILITY 1. The minimum volume required is 250 µL of specimen for the first test, and 25 µL more for each additional test. 2. Mix thawed specimens thoroughly by low speed vortexing or by inverting 10 times. Visually inspect the specimens. If layering or stratification is observed, continue mixing until specimens are visibly homogeneous. 3. Samples having particulate matter, turbidity, lipemia or erythrocyte debris may require clarification by filtration or centrifugation before testing. Check for and remove air bubbles before assaying. 1. Frozen (-20˚C or below). Mix thawed samples well before testing. 2. Specimens may be stored in glass or plastic vials. No additives or preservatives are required to maintain integrity of the samples. 3. Avoid multiple freeze/thaw cycles. Samples should not be tested if exposed to more than three freeze/thaw cycles. After separation from cells: • Ambient: 3 days • Refrigerated: 1 week • Frozen: 6 months V. EQUIPMENT, REAGENTS AND SUPPLIES TEST INSTRUMENT: DiaSorin LIAISON System MATERIALS PROVIDED DiaSorin LIAISON Reagent Integral (Cat. No. 310600) MATERIALS REQUIRED BUT NOT PROVIDED 1. DiaSorin LIAISON System (including sample racks). eterr:Library:Mail Downloads:25OHVitDTotal.doc Page 3 of 15
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25OH Vitamin D Total Assay - University of California, San Francisco

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