Which laboratory test is recommended to check vitamin D (25-hydroxyvitamin D) levels?

Which Laboratory Test to Check Vitamin D Levels

Order a serum total 25-hydroxyvitamin D [25(OH)D] test that measures both 25(OH)D2 and 25(OH)D3 forms. 1

The Standard Test

• Serum total 25(OH)D is the gold standard for assessing vitamin D status, as it accurately reflects total body vitamin D stores and availability 1
• The assay must measure both 25(OH)D2 (ergocalciferol) and 25(OH)D3 (cholecalciferol) forms, as both contribute to total vitamin D status 2, 1
• This is particularly critical in countries where both vitamin D2 and D3 supplements are available, as high-dose prescription preparations often contain D2 2

Sample Collection Specifications

• Use serum as the sample of choice 2, 1
• Avoid gel tubes until more data are available, as they can interfere with LC-MS/MS measurements 2
• Li-heparin plasma is acceptable if serum is not available 3

Available Testing Methods

The following methods can measure 25(OH)D, listed in order of preference:

• Liquid chromatography-tandem mass spectrometry (LC-MS/MS): Offers superior precision and flexibility, gaining ascendance as the preferred method 1, 4
• High-performance liquid chromatography (HPLC): Reliable method for measuring 25(OH)D levels 1, 5
• Immunoassays (enzyme-immunosorbent, immunochemiluminescence, immunofluorescence): Widely available but show greater variability 6, 7

Critical Quality Considerations

Verify your laboratory participates in standardization programs to ensure accurate results:

• The Vitamin D Standardization Program (VDSP) 1
• CDC's Vitamin D Standardization Certification Program 1
• External quality control schemes like UK-based DEQAS 2

Common Pitfalls to Avoid

• Assay variability is substantial: Different methods can vary by 10-20%, and classification of samples as "deficient" versus "nondeficient" can vary by 4-32% depending on which assay is used 1
• Don't rely on assays that measure only one form: Some immunoassays underdetect 25(OH)D2, measuring only about 55% of the actual concentration compared to LC-MS/MS 3
• Avoid population-based reference values: These are confusing for clinicians; use recommended target ranges instead 2

Result Interpretation

• Target optimal range: 30-40 ng/mL minimum, particularly for patients with musculoskeletal problems, cardiovascular disease, autoimmune disease, or cancer 1
• Upper safety limit: 100 ng/mL 1
• Express results in ng/mL for consistency 2

Special Population Considerations

• African Americans: Total serum 25(OH)D may underestimate vitamin D status due to differences in vitamin D binding protein, though bioavailable 25(OH)D may be similar across races 1
• Patients on vitamin D2 supplementation: Ensure your assay accurately measures 25(OH)D2, as many immunoassays significantly underestimate this form 3

Timing of Testing

• Baseline measurement: Perform before starting supplementation (except in high-risk groups like institutionalized individuals who can start supplementation without testing) 2
• Follow-up testing: Wait at least 3 months after starting supplementation to allow plateau levels to be reached 2, 1