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Vitamin D Testing

The Mayo Medical Laboratories Difference


February 2009

Ligand-Binding Assays

Technical aspects of ligand-binding assays (ie, radioimmunoassay, competitive protein-binding assays) include:

  • Fundamentally difficult because such assays work best in an aqueous environment and 25OH Vit D2 and 25OH Vit D3 are poorly soluble in water.
    • Manual extraction assays can overcome solubility problems, but suffer from increased imprecision.
  • Lack of result comparability between different assays, and often even between the same assays performed in different laboratories.5
    • One comparison of six laboratories found that “whether a patient is considered as being vitamin D insufficient depends, in large measure, on the laboratory used.”4
  • Some assays significantly overestimate vitamin D levels, others significantly underestimate them.
    • A study conducted by the Vitamin D External Quality Assessment Scheme (DEQAS) showed a 31% overestimation by one immunoassay method.6
    • More typically, underestimation is observed. This is particularly the case in patients on vitamin D2 therapy; some immunoassays cannot detect 25 OH Vit D2.
  • Ligand-binding assays cannot distinguish between 25OH VitD2 and 25OH VitD3. This makes it difficult to judge whether patients are compliant with therapies or suffer from vitamin D malabsorption.

High-performance liquid chromatography (HPLC)

Technical aspects of HPLC include:

  • Overcomes most of the problems of the ligand-binding assays, and it was previously seen as the “gold standard.”
  • Requires a relatively large sample volume (≥1.0 mL).
  • Labor-intensive and time-consuming

Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Technical aspects of LC-MS/MS include:

  • Accurate and precise, due to internal standard and physico-chemical methodology.
    • “Gold standard” method.
    • Uses standards of defined concentrations.
    • Facilitates lab-to-lab and assay-to-assay comparisons with other MS/MS and HPLC assays.
    • Results may be used to determine if target therapeutic levels (25-80 ng/mL) have been obtained.
  • Not subject to lot-to-lot variation, reagent availability issues, or licensing fees associated with proprietary kits.
  • Faster than HPLC.
  • Separates 25OH VitD2 and 25OH VitD3. In patients who show no improvement on replacement therapy (VitD2), absence of rise in 25OH VitD2 may indicate noncompliance or malabsorption, whereas rise in 25OH VitD2 and total vitamin D levels may indicate vitamin D resistance.
  • Uses a smaller specimen volume than HPLC (0.25 mL).

How Does the Mayo Test Differ?

The Mayo Vitamin D deficiency test offers key differentiators:

  • Mayo-developed and Mayo-validated LC-MS/MS assay.
  • Requires fewer repeat analyses as a result of a wider reportable range.
  • Provides 25OH VitD2 and 25 Vit D3 levels in addition to the total vitamin D level. This allows assessment of the source of the deficiency and also facilitates treatment monitoring.
  • The total 25OH Vit D measured with the new assay correlates well with our previous methodology, which in itself has been shown to outperform other laboratories and tests.
  • Improves on accuracy and precision of ligand assays, including our previous assay: ~10% CV at low 25OH Vit D levels (10 ng/mL)7

Key