Vitamin D Testing: Measuring 25-Hydroxyvitamin D Levels
The standard laboratory test to measure vitamin D levels is the total serum 25-hydroxyvitamin D [25(OH)D] test, which measures both 25(OH)D2 and 25(OH)D3 forms. 1, 2
Understanding Vitamin D Testing
- 25-hydroxyvitamin D [25(OH)D] is the primary circulating form of vitamin D and serves as the best marker of overall vitamin D status 2
- Testing should measure both 25(OH)D2 (ergocalciferol) and 25(OH)D3 (cholecalciferol) forms, as both contribute to total vitamin D status 1
- 1,25-dihydroxyvitamin D [1,25(OH)2D] is NOT recommended for routine vitamin D status assessment, as it provides no reliable information about vitamin D status and may even be normal or elevated in vitamin D deficiency due to secondary hyperparathyroidism 2, 3
Testing Methods
Several methods are available to measure 25(OH)D levels:
- Immunoassays (automated, widely available) 4
- High-performance liquid chromatography (HPLC) 1
- Combined high-performance liquid chromatography and mass spectrometry (LC-MS/MS) 1, 4
- Competitive protein binding assays 1
Important Considerations for Testing
- Choose an assay that measures both 25(OH)D2 and 25(OH)D3 forms, especially in countries where both vitamin D2 and D3 supplements are available 1
- Be aware that variability between assay methods and between laboratories using the same methods may range from 10% to 20% 1
- Classification of samples as "deficient" or "nondeficient" can vary by 4% to 32% depending on which assay is used 1
- Serum is the sample of choice; avoid gel tubes until more data are available 1
Interpretation of Results
- Most experts consider 25(OH)D levels <20 ng/mL as vitamin D deficiency 2
- 25(OH)D levels of 21-29 ng/mL are generally considered insufficient 2
- Target range for optimal health should be at least 30-40 ng/mL, particularly in patients with or at risk for musculoskeletal problems, cardiovascular disease, autoimmune disease, and cancer 1
- Upper safety limit is generally considered to be 100 ng/mL 1
Monitoring After Supplementation
- When monitoring response to vitamin D supplementation, wait at least 3 months after starting treatment before retesting 25(OH)D levels 1
- Response to treatment varies based on environmental factors and baseline 25(OH)D levels 1
Special Considerations
- Free 25(OH)D testing may be more accurate in certain conditions affecting vitamin D binding protein (DBP) or albumin levels, such as liver disease, pregnancy, or kidney disease, but is not routinely available in clinical practice 5
- Dried blood spot testing is emerging as a convenient alternative to serum testing and may be valuable for large-scale screening 6
Standardization Efforts
- Several organizations are working to improve testing accuracy, including the Vitamin D Standardization Program and the Centers for Disease Control and Prevention's Vitamin D Standardization Certification Program 1
- Despite these efforts, significant differences remain among various methods and laboratories 4