What is the recommended test to order for serum vitamin D (25-hydroxyvitamin D) level testing?

Recommended Test for Serum Vitamin D Level Testing

For optimal clinical care, the recommended test for serum vitamin D level testing is an assay that measures both 25-hydroxyvitamin D2 (25(OH)D2) and 25-hydroxyvitamin D3 (25(OH)D3), with results reported as the total 25(OH)D concentration. 1

Rationale for Testing Total 25(OH)D

• 25-hydroxyvitamin D (25(OH)D) is the primary circulating form of vitamin D and is considered the best marker of overall vitamin D status 2
• Both forms (25(OH)D2 and 25(OH)D3) contribute to vitamin D status and should be measured together 1
• Using an assay that measures both forms is particularly important in countries where both vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol) are available for supplementation 1
• In some countries, high-dose vitamin D preparations available by prescription contain the D2 form, making measurement of both forms essential 1

Testing Methodology Considerations

• Common testing methods include:
  • Immunoassays (automated)
  • High-performance liquid chromatography (HPLC)
  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) 3, 2
• LC-MS/MS is often considered the reference method, but properly standardized immunoassays are acceptable for clinical practice 3
• Laboratories should participate in external quality control programs (such as DEQAS) to ensure accuracy 1
• Internal quality control programs should be implemented with defined criteria for accepting or rejecting analytical runs 1

Sample Collection Guidelines

• Serum is the recommended sample type for 25(OH)D testing 1
• Until more data are available, gel tubes should be avoided as they may interfere with some measurement methods, particularly LC-MS/MS 1
• 25(OH)D is very stable in serum, making it suitable for routine clinical testing 1

Interpretation of Results

• The expert panel recommends that 25(OH)D levels should be above 30 ng/mL (75 nmol/L) for optimal health benefits 1
• An upper safety limit of 100 ng/mL (250 nmol/L) is recommended 1
• Results should be expressed in ng/mL (or nmol/L with clear conversion factors) 1
• Laboratory reference ranges should align with clinical guidelines rather than population-based reference values 1

Clinical Pitfalls to Avoid

• Variability between assay methods and laboratories may range from 10% to 20%, which can affect clinical decision-making 1
• Classification of samples as "deficient" or "non-deficient" may vary by 4% to 32% depending on the assay used 1
• 25(OH)D may act as a negative acute-phase reactant, with levels decreasing during inflammation 1
• Measurement uncertainty must be considered when interpreting results near clinical decision thresholds 1
• Some assays may underestimate 25(OH)D2 compared to 25(OH)D3, potentially leading to underestimation of total vitamin D status in patients taking vitamin D2 supplements 4

By ordering a total 25(OH)D assay that measures both 25(OH)D2 and 25(OH)D3, clinicians can accurately assess vitamin D status and make appropriate treatment decisions to optimize patient outcomes related to musculoskeletal health, cardiovascular disease, immune function, and potentially cancer risk.