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Vitamin D, 25-Hydroxy, Total, Immunoassay

Vitamin D, 25-Hydroxy, Total, Immunoassay

Test Summary

Vitamin D, 25-Hydroxy, Total, Immunoassay


Clinical Use

  • Diagnose vitamin D deficiency or toxicity

  • Monitor response to vitamin D2 or vitamin D3 supplementation

Clinical Background

As much as 68% to 77% of the population is estimated to have suboptimal (<30 ng/mL) levels of vitamin D.1,2 This important nutrient promotes skeletal health by enhancing the intestinal absorption of calcium and phosphorus. Deficiency is associated with bone diseases such as rickets, osteomalacia, and osteoporosis. Emerging evidence also suggests links to nonskeletal illnesses such as cancer (especially colorectal and breast), cardiovascular disease, and infectious and autoimmune diseases.3,4 Vitamin D toxicity, which manifests as hypercalcemia, hypercalciuria, or kidney stones, is rare but can result at serum levels above 150 ng/mL.4 Current guidelines recommend maintaining adequate vitamin D levels (with vitamin D supplementation if necessary) to maximize bone health and prevent bone disease.4-6 Accurate determination of vitamin D levels can help diagnose vitamin D deficiency and toxicity, and inform clinical management.

The term "vitamin D" typically refers to 2 molecular forms: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Vitamin D3 is the main form and is produced in the skin in response to sunlight.4 However, both forms can be obtained in relatively small amounts through normal diet. Both are also available as over-the-counter supplements and as high-dose prescription formulations in the United States.3 Vitamin D3 is also available as a high-dose supplement.

Vitamins D2 and D3 are metabolized to produce different 25-hydroxyvitamin D (25[OH]D) forms: 25(OH)D2 and 25(OH)D3; these metabolites are then converted to their corresponding active forms.4 The sum of 25(OH)D2 and 25(OH)D3 concentrations yields the total 25(OH)D level, which is the accepted standard for determining vitamin D status.4 The active metabolite of vitamin D (1,25-dihydroxyvitamin D) is not useful for assessing vitamin D status because its levels are often normal or even elevated in patients with vitamin D deficiency.4

Quest Diagnostics offers the Vitamin D, 25-Hydroxy, Total, Immunoassay test (test code: 17306), which uses a chemiluminescent method to assess 25(OH)D status in serum. The method is standardized to the National Institute of Standards and Technology (NIST) standard reference material (SRM) 972, ensuring its accuracy. Furthermore, the test has also been certified by the Centers for Disease Control and Prevention (CDC) Vitamin D Standardization Certification Program, demonstrating precision and lack of bias in the method as compared to the CDC's LC/MS/MS reference method.

Individuals Suitable for Testing

  • Individuals with suspected vitamin D deficiency (eg, those with osteoporosis, osteomalacia, or persistent, nonspecific musculoskeletal pain)

  • Individuals at increased risk for vitamin D deficiency (eg, pregnant, lactating, dark-skinned, elderly, obese, or housebound; those dwelling in latitudes higher than 33° north or 33° south; and infants who are breastfeeding without vitamin D supplementation or from vitamin D-deficient mothers)

  • Individuals with disorders associated with reduced 25(OH)D levels (see Table)

  • Individuals being treated with vitamin D2 or vitamin D3 supplementation

  • Individuals with suspected toxicity (eg, those with hypercalcemia of obscure origin)


  • Immunoassay:

    –  Separation of serum 25(OH)D from its binding protein

    –  Competitive binding of serum 25(OH)D and tracer-labeled 25(OH)D to specific antibody

    –  Detection and quantitation via chemiluminescence reaction

  • Analytical sensitivity: 4 ng/mL
  • Analytical specificity: 100% cross-reactivity with 25(OH)D2 and 25(OH)D3; 1.9% cross-reactivity with vitamin D2 and D3; 6.7% and 9.3% cross-reactivity with 1,25-dihydroxyvitamin D2 and D3, respectively; and 1.3% cross-reactivity with 3-epi-25(OH)D3
  • Reportable range: 4-150 ng/mL

For the LC/MS/MS method, use test code 92888 (children and adults) or 91935 (infants <3 years).

Interpretive Information

Abnormal 25(OH)D levels are associated with a range of conditions and medications (Table).3,4,7-16 There is no consensus about the optimal total 25(OH)D level, which may vary with the assay used and functional outcome measured. However, many experts accept a minimum of 30 ng/mL with a range of 40 to 60 ng/mL as optimal.3 A level up to 100 ng/mL is considered to be safe.3,4 Total 25(OH)D levels ≤20 ng/mL suggest vitamin D deficiency, while levels between 21 and 29 ng/mL suggest insufficiency.4 Expert opinions also vary about what constitutes 25(OH)D toxicity, with reported thresholds ranging from 50 ng/mL for outcome measures such as increased risk for mortality not related to calcium and bone metabolism (125 nmol/L)1 to 150 ng/mL (374 nmol/L) for mortality related to calcium and bone metabolism.

Table. Effect of Various Disorders and Medications on 25-Hydroxyvitamin D Concentration3,4,7-16


25(OH)D Concentration

Chronic kidney disease3,7

↓ or N

Fat malabsorption disorders, short bowel syndrome, inflammatory bowel disease, Crohn disease4,8

Hypercalcemia of cancera,9

↓ or N or ↑

Hypophosphatemic ricketsb,3


Lymphoma, granulomatous disorders3,4

Medications that increase vitamin D metabolism (eg, anticonvulsants, antiretrovirals, and glucocorticoids)3,4

Nephrotic syndrome10

Nutritional rickets or osteomalacia11



↓or N

Primary hyperparathyroidism3,4

↓ or N

Secondary hyperparathyroidism13

Severe parenchymal liver disease (impaired 25-hydroxylation of vitamin D)10,14

Tumor-induced osteomalacia (oncogenic osteomalacia)3,15


Vitamin D-dependent rickets, type Ic,16


Vitamin D-dependent rickets, type IId,16


Vitamin D (25[OH]D) toxicity (uncommon)10

25(OH)D, 25-hydroxyvitamin D; N, normal; ↑, elevated; ↓, reduced.


PTHrP (parathyroid hormone–related peptide)-mediated


Autosomal dominant or X-linked hypophosphatemic rickets


Also called pseudo-vitamin D-deficiency rickets.


Also called hereditary vitamin D-resistant rickets.


  1. Lai JK, Lucas RM, Clements MS, et al. Assessing vitamin D status: pitfalls for the unwary. Mol Nutr Food Res. 2010;54:1062-1071.

  2. Ginde AA, Liu MC, Camargo CA, Jr. Demographic differences and trends of vitamin D insufficiency in the US population, 1988-2004. Arch Intern Med. 2009;169:626-632.

  3. Holick MF. Vitamin D deficiency. N Engl J Med. 2007;357:266-281.

  4. Holick MF, Binkley NC, Bischoff-Ferrari HA, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2011;96:1911-1930.

  5. Ross AC, Manson JE, Abrams SA, et al. The 2011 report on dietary reference intakes for calcium and vitamin D from the Institute of Medicine: what clinicians need to know. J Clin Endocrinol Metab. 2011;96:53-58.

  6. Watts NB, Bilezikian JP, Camacho PM, et al. American Association of Clinical Endocrinologists medical guidelines for clinical practice for the diagnosis and treatment of postmenopausal osteoporosis. Endocr Pract. 2010;16(suppl 3):1-37.

  7. National Kidney Foundation. K/DOQI clinical practice guidelines for bone metabolism and disease in chronic kidney disease. Am J Kidney Dis. 2003;42(suppl 3):S1-S201.

  8. Margulies SL, Kurian D, Elliott MS, et al. Vitamin D deficiency in patients with intestinal malabsorption syndromes—think in and outside the gut. J Dig Dis. 2015;16:617-633.

  9. Goldner W. Cancer-related hypercalcemia. J Oncol Pract. 2016;12:426-432.

  10. Bouillon R. Vitamin D: from photosynthesis, metabolism, and action to clinical applications. In: Jameson JL, De Groot LJ, eds. Endocrinology. 6th ed. Philadelphia, PA: Saunders; 2010:1089-1110.

  11. Pettifor JM. Calcium and vitamin D metabolism in children in developing countries. Ann Nutr Metab. 2014;64(suppl 2):15-22.

  12. Lips P. Relative value of 25(OH)D and 1,25(OH)2D measurements. J Bone Miner Res. 2007;22:1668-1671.

  13. Lips P. Vitamin D deficiency and secondary hyperparathyroidism in the elderly: consequences for bone loss and fractures and therapeutic implications. Endocr Rev. 2001;22:477-501.

  14. Stokes CS, Volmer DA, Grunhage F, et al. Vitamin D in chronic liver disease. Liver Int. 2013;33:338-352.

  15. Hautmann AH, Hautmann MG, Kölbl O, et al. Tumor-induced osteomalacia: an up-to-date review. Curr Rheumatol Rep. 2015;17:512.

  16. St-Arnaud R, Glorieux FH. Genetic defects in vitamin D metabolism and action. In: Jameson JL, De Groot LJ, eds. Endocrinology. 6th ed. Philadelphia, PA: Saunders; 2010:1236-1249.

Content reviewed 07/2018

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