Which Vitamin D Level Should Be Ordered
Order serum 25-hydroxyvitamin D [25(OH)D] to assess vitamin D status, ensuring the assay measures both 25(OH)D2 and 25(OH)D3 forms. 1
The Standard Test
Serum 25-hydroxyvitamin D is the barometer for vitamin D status and represents the most reliable biochemical marker for defining vitamin D stores in the body. 2
The test must measure total 25(OH)D, which includes both the D2 form (ergocalciferol) and D3 form (cholecalciferol), as both contribute to overall vitamin D status. 1
Do not order 1,25-dihydroxyvitamin D [1,25(OH)₂D] for assessing vitamin D status—this active form provides no information about body stores and is often normal or even elevated in vitamin D deficiency due to secondary hyperparathyroidism. 3
Why 25(OH)D Is the Correct Test
25(OH)D has a longer half-life (approximately 2-3 weeks) compared to the active form, making it a stable indicator of vitamin D status over time. 4
Serum 25(OH)D levels directly reflect both dietary intake and cutaneous synthesis of vitamin D, providing an accurate picture of total body vitamin D availability. 3
The measurement of 25(OH)D is recognized across all major guidelines as the gold standard for vitamin D assessment. 2, 1
Assay Selection Considerations
Choose an assay that measures both 25(OH)D2 and 25(OH)D3, particularly important in regions where both vitamin D2 and D3 supplements are available. 1
Available methods include high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and competitive protein binding assays. 1
Use serum as the sample of choice and avoid gel tubes until more data are available on their reliability. 1
Important Caveats About Testing Accuracy
Be aware that variability between assay methods and between laboratories using the same method can range from 10% to 20%. 1
Classification of samples as "deficient" versus "nondeficient" can vary by 4% to 32% depending on which assay is used, highlighting the importance of using standardized, calibrated assays. 1
Several organizations, including the Vitamin D Standardization Program and CDC's Vitamin D Standardization Certification Program, are working to improve testing accuracy across laboratories. 1
When to Retest After Supplementation
Wait at least 3 months after initiating vitamin D supplementation before rechecking 25(OH)D levels, as response to treatment varies based on environmental factors and baseline levels. 1, 5
For patients with chronic kidney disease on vitamin D therapy, measure serum calcium and phosphorus every 3 months in addition to periodic 25(OH)D monitoring. 2