What is the best method to measure serum vitamin D (25(OH)D) levels in adults, particularly those at risk for deficiency?

Best Method to Measure Serum Vitamin D Levels

Measure total serum 25-hydroxyvitamin D [25(OH)D] using an assay that detects both 25(OH)D2 and 25(OH)D3 forms, with liquid chromatography-tandem mass spectrometry (LC-MS/MS) being the gold standard methodology. 1, 2

Recommended Testing Methodology

Primary Assay Selection

• Choose an assay that measures both 25(OH)D2 (ergocalciferol) and 25(OH)D3 (cholecalciferol) since both forms contribute to total vitamin D status, particularly in countries where both supplement types are available. 3, 1
• LC-MS/MS has become the gold standard with reference method procedures accepted by the Joint Committee for Traceability in Laboratory Medicine. 2
• Alternative methods include high-performance liquid chromatography (HPLC) and competitive protein binding assays, though these have varying accuracy. 1

Critical Quality Assurance Requirements

• Verify that your laboratory participates in the CDC's Vitamin D Standardization Certification Program to ensure quality and comparability of results. 3, 1
• Laboratories should participate in external quality control schemes that provide materials with documented commutability with human sera (such as DEQAS). 3
• Internal quality control programs using different level samples must be carried out in every laboratory. 3

Important Testing Considerations

Sample Collection

• Use serum as the sample of choice and avoid gel tubes until more data are available, as gel tubes can interfere with LC-MS/MS measurements. 3, 1
• 25(OH)D is very stable in serum, making it an ideal marker for vitamin D status. 3

Assay Variability Issues

• Be aware that classification of samples as "deficient" or "nondeficient" can vary by 4% to 32% depending on which assay is used. 3, 1, 2
• Testing variability between methods and between laboratories using the same method ranges from 10% to 20%. 3, 2
• The National Institute of Standards and Technology standard reference materials improve accuracy for HPLC and mass spectrometry but have limited effects on immunoassay methods. 3

Reporting Requirements

• If using an assay that separates 25(OH)D2 and 25(OH)D3 (such as HPLC or LC-MS/MS), report the sum of both compounds as the main result. 3
• This is particularly important since an increasing number of tested serum samples contain significant amounts of 25(OH)D2, with 3% containing concentrations >20 ng/mL in some populations. 3

Common Pitfalls to Avoid

Heterophilic Antibody Interference

• Heterophilic antibodies can bind to assay components in immunoassay-based methods, causing falsely elevated or decreased measurements with variations of 4-32% in sample classification. 1
• This interference is more problematic with immunoassays than with LC-MS/MS methods. 1

Race-Based Interpretation

• Total serum 25(OH)D may underestimate vitamin D status in African Americans due to differences in vitamin D binding protein, though bioavailable 25(OH)D may be similar across races. 3, 2
• However, commercial testing of bioavailable 25(OH)D is not currently available, so total 25(OH)D remains the standard. 3

Timing of Measurement

• When monitoring response to supplementation, wait at least 3 months before retesting to allow plateau levels to be reached with daily dosing. 3, 1
• For intermittent regimens, measure 25(OH)D at least 3 months after starting, just prior to the next dose administration. 3

Why 25(OH)D is the Preferred Marker

• 25(OH)D is the most reliable biochemical marker for defining vitamin D stores because it represents the sum of dietary intake and sunlight-induced production. 1, 4, 5
• It has a longer half-life than 1,25-dihydroxyvitamin D and better reflects overall vitamin D status. 4, 5
• Serum vitamin D itself is less useful clinically, serving mainly to determine sunlight contribution or assess absorption in malabsorption syndromes. 4