Vitamin D Blood Tests
The Standard Test
The total serum 25-hydroxyvitamin D [25(OH)D] test is the single best and only routinely recommended blood test for assessing vitamin D status. 1, 2 This test measures both 25(OH)D2 (ergocalciferol, from plant sources and supplements) and 25(OH)D3 (cholecalciferol, from animal sources, supplements, and skin synthesis), which together reflect your total vitamin D stores. 1, 3
Available Testing Methods
The 25(OH)D test can be performed using several different laboratory techniques, each with distinct characteristics:
Gold Standard Method
- Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is now considered the gold standard methodology with the highest accuracy and has been accepted by the Joint Committee for Traceability in Laboratory Medicine. 4, 5
Alternative Methods
- High-performance liquid chromatography (HPLC) can measure 25(OH)D levels with good accuracy. 1
- Immunoassays (including radioimmunoassay and ELISA) are widely used automated methods but show more variability than mass spectrometry. 5, 6
- Competitive protein binding assays are also available but less commonly used. 1
Critical Testing Considerations
Method Variability Is a Major Problem
- Classification of your sample as "deficient" or "nondeficient" can vary by 4% to 32% depending on which assay the laboratory uses. 7, 1, 4
- Testing variability between different methods and even between laboratories using the same method ranges from 10% to 20%. 1, 4
- This means you could be classified as deficient at one lab and normal at another using the same blood sample. 1
Ensure Your Lab Uses a Quality-Assured Method
- Verify that your laboratory participates in the CDC's Vitamin D Standardization Certification Program, which helps maintain quality and comparability of results. 1, 4
- Choose an assay that measures both 25(OH)D2 and 25(OH)D3 forms, especially if you take supplements (since both D2 and D3 supplements are available). 1
- Use serum samples and avoid gel tubes until more data are available on their effects. 1
Other Vitamin D Tests (Not Routinely Recommended)
1,25-Dihydroxyvitamin D Test
- This measures the most biologically active form of vitamin D (calcitriol), but it should NOT be used to assess vitamin D status. 3, 2
- This test has limited clinical utility and is not part of standard vitamin D testing. 2
- It may be ordered in specific disease management scenarios (kidney disease, certain bone disorders) but not for general screening. 1
Free 25(OH)D Test
- This measures only the unbound, "free" vitamin D rather than total vitamin D. 8
- Most vitamin D circulates bound to vitamin D-binding protein (DBP) and albumin, with only tiny amounts free. 8
- Total 25(OH)D may underestimate vitamin D status in African Americans due to differences in vitamin D binding protein, although bioavailable 25(OH)D may be similar across races. 1
- Free 25(OH)D testing may be more accurate in patients with liver disease, kidney disease with proteinuria, pregnancy, or conditions affecting DBP or albumin levels, but this test is not widely available or standardized. 8
When to Retest After Supplementation
- Wait at least 3 months after starting vitamin D supplementation before retesting 25(OH)D levels to allow adequate time for levels to stabilize. 1
- Response to treatment varies based on environmental factors and your baseline 25(OH)D levels. 1
Common Pitfalls to Avoid
- Don't order 1,25-dihydroxyvitamin D when you want to check vitamin D status—this is a common error that provides no useful information for assessing deficiency. 2
- Don't assume all 25(OH)D tests are equivalent—significant differences exist between methods and laboratories. 7, 5
- Don't interpret results without knowing which assay was used, as cutoffs for "deficiency" may need adjustment based on the method. 1
- Don't use gel tubes for sample collection until more data confirm they don't interfere with results. 1