Best Test for Vitamin D3 Measurement
The serum 25-hydroxyvitamin D [25(OH)D] test is the single best and only test you should order to assess vitamin D status, and it must measure both 25(OH)D2 and 25(OH)D3 forms. 1, 2, 3, 4
Why 25(OH)D is the Gold Standard
Serum 25(OH)D is the most reliable biochemical marker for defining total body vitamin D stores because it has a longer half-life (approximately 2-3 weeks) and reflects both dietary intake and cutaneous synthesis. 2, 3, 4 This metabolite circulates at concentrations 1000-fold higher than the active form 1,25-dihydroxyvitamin D, making it easier to measure accurately. 3, 4
Do Not Order 1,25-Dihydroxyvitamin D
The active metabolite 1,25(OH)2D should not be used to assess vitamin D status because it is tightly regulated by parathyroid hormone and does not reflect body stores—patients can have normal or even elevated 1,25(OH)2D levels despite severe vitamin D deficiency. 2, 4 The need for measuring serum 1,25(OH)2D is limited to specific conditions like hypercalcemia workup or suspected vitamin D receptor defects, not routine deficiency screening. 4
Critical Assay Requirements
Must Measure Both D2 and D3 Forms
Your laboratory assay must measure total 25(OH)D, meaning both 25(OH)D2 (ergocalciferol) and 25(OH)D3 (cholecalciferol) combined. 1, 2 This is essential because patients may be taking either vitamin D2 or D3 supplements, and both contribute to total vitamin D status. 1, 2 Some older assays only measure one form, which can lead to significant underestimation of true vitamin D status. 2
Preferred Laboratory Methods
The most accurate methods for measuring 25(OH)D are: 2, 5, 4
- Liquid chromatography-tandem mass spectrometry (LC-MS/MS): This is the gold standard method with superior accuracy and ability to separately quantify D2 and D3 forms. 2, 5, 4
- High-performance liquid chromatography (HPLC): Also highly accurate when standardized properly. 2, 3
Automated immunoassays are widely used but have greater variability between methods—classification of samples as "deficient" versus "nondeficient" can vary by 4-32% depending on which immunoassay is used. 1, 2, 5 If your laboratory uses immunoassays, verify they participate in the CDC's Vitamin D Standardization Certification Program. 1, 2
Important Testing Considerations
Sample Collection
Use serum as the sample of choice; avoid gel separator tubes until more data are available on their impact on measurement accuracy. 2 The assay should be performed on fasting or non-fasting samples—food intake does not significantly affect 25(OH)D levels since it reflects long-term status. 2
Timing of Testing
For patients at risk (elderly, dark-skinned individuals, those with limited sun exposure, kidney disease, liver disease, malabsorption), test 25(OH)D levels before initiating supplementation to establish baseline status. 1, 6 However, for certain high-risk groups—dark-skinned or veiled individuals with minimal sun exposure, elderly and institutionalized individuals—you can start supplementation with 800 IU daily without baseline testing. 1, 6
When to Retest After Supplementation
Wait at least 3 months after starting vitamin D supplementation before rechecking 25(OH)D levels because vitamin D has a long half-life and serum concentrations need adequate time to reach plateau and reflect true response to therapy. 1, 6, 2, 7 Testing earlier will not accurately reflect steady-state levels and may lead to inappropriate dose adjustments. 7
If using intermittent dosing regimens (weekly or monthly), measure levels just prior to the next scheduled dose. 6, 7
Interpretation of Results
Target Levels
Aim for serum 25(OH)D levels of at least 30 ng/mL (75 nmol/L) for optimal health benefits, particularly for fracture prevention and fall reduction in at-risk populations. 1, 6, 2 The expert consensus defines: 1, 6, 2
- Deficiency: <20 ng/mL (<50 nmol/L)
- Insufficiency: 20-30 ng/mL (50-75 nmol/L)
- Optimal: ≥30 ng/mL (≥75 nmol/L)
- Upper safety limit: 100 ng/mL (250 nmol/L)
Assay Variability Caveat
Be aware that variability between different assay methods and between laboratories using the same method can range from 10-20%, which may affect clinical decision-making at borderline values. 1, 2, 5 When monitoring a patient over time, use the same laboratory and same assay method when possible. 2, 5
Special Consideration for African Americans
Total serum 25(OH)D may underestimate vitamin D status in African Americans due to differences in vitamin D binding protein genetics, though bioavailable 25(OH)D concentrations may be similar to other racial groups. 1 However, commercial testing for bioavailable 25(OH)D is not currently widely available, so continue using total 25(OH)D with awareness of this limitation. 1
Common Pitfalls to Avoid
- Do not order 1,25-dihydroxyvitamin D for routine vitamin D status assessment—it does not reflect body stores. 2, 4
- Do not retest before 3 months after starting supplementation—levels need time to stabilize. 1, 6, 7
- Do not use assays that only measure 25(OH)D3—you will miss patients taking vitamin D2 supplements. 1, 2
- Do not ignore laboratory method differences—verify your lab uses a standardized, validated assay that measures both D2 and D3. 1, 2, 5
- Account for seasonal variation—levels are typically lowest in late winter/early spring and highest in late summer/early fall. 7