How to Test for Vitamin D in the Blood
Order a serum 25-hydroxyvitamin D [25(OH)D] test using a reliable assay method—this is the gold standard for assessing vitamin D status. Do not order a 1,25-dihydroxyvitamin D test for routine vitamin D assessment, as it does not reflect vitamin D stores 1.
Which Test to Order
- Order: Serum 25(OH)D (also called 25-hydroxyvitamin D or calcidiol)
- Do NOT order: 1,25(OH)₂D (calcitriol) for routine vitamin D status assessment
The 25(OH)D is the major circulating form with a half-life of 2-3 weeks and accurately reflects total body vitamin D stores 1. In contrast, 1,25(OH)₂D has a 4-hour half-life, circulates at 1000 times lower concentration, and is tightly regulated by PTH, calcium, and phosphate—making it frequently normal or even elevated in vitamin D deficiency due to secondary hyperparathyroidism 1.
Testing Methods Available
Modern laboratories use two primary approaches 2, 3:
- Automated immunoassays (e.g., Elecsys, Liaison): Faster, high-throughput, suitable for routine clinical use
- Liquid chromatography-tandem mass spectrometry (LC-MS/MS): Greater precision and flexibility, increasingly preferred as the reference standard
Both methods are adequate for clinical care when standardized using National Institute of Standards and Technology (NIST) reference materials 1, 4. The key is using a laboratory that participates in standardization programs to ensure accuracy.
Interpreting Results
Use these thresholds when results return 1:
- Deficiency: <20 ng/mL (<50 nmol/L)
- Insufficiency: 21-29 ng/mL (52.5-72.5 nmol/L)
- Sufficiency: ≥30 ng/mL (≥75 nmol/L)
- Toxicity risk: >100 ng/mL (>250 nmol/L)
Who Should Be Tested
Do not screen asymptomatic, low-risk adults—the USPSTF found insufficient evidence to support population-wide screening 5.
Test selectively in high-risk groups where prompt intervention would be beneficial 1:
- Age >65 years
- Pregnant women
- Patients with recurrent falls or fragility fractures
- Osteoporosis or secondary hyperparathyroidism
- Chronic kidney disease
- Malabsorption syndromes
- Obesity
- Dark-skinned individuals at higher latitudes
- Those taking medications affecting vitamin D metabolism
Important Caveats
Assay variability exists despite standardization efforts—different methods and laboratories can produce different results for the same sample 2, 6. When targeting a specific threshold, consider aiming for values well above the cutoff (e.g., 40 ng/mL ensures the true value exceeds 30 ng/mL) to account for measurement uncertainty 1.
Sample type: Both serum and heparinized plasma are acceptable and show equivalent results 6.
When to use 1,25(OH)₂D testing: Reserve this only for monitoring specific conditions like chronic kidney disease, hereditary phosphate-losing disorders, oncogenic osteomalacia, pseudovitamin D-deficiency rickets, sarcoidosis, or lymphomas 1.