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Vitamin D Testing

The Mayo Medical Laboratories Difference

Updated: September 2013
Published: 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 25-OH-VitD2 and 25-OH-VitD3 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 laboratories5
    • A comparison of 6 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 method6
    • More typically, underestimation is observed. This is particularly the case in patients on vitamin D2 therapy; some immunoassays cannot detect 25-OH-VitD2.
  • Ligand-binding assays cannot distinguish between 25-OH-VitD2 and 25-OH-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 physicochemical 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 25-OH-VitD2 and 25-OH-VitD3. In patients who show no improvement on replacement therapy (VitD2), absence of rise in 25-OH-VitD2 may indicate noncompliance or malabsorption, whereas rise in 25-OH-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 Clinic Test Differ?

The Mayo Clinic vitamin D deficiency test offers key differentiators:

  • Mayo Clinic-developed and Mayo Clinic-validated LC-MS/MS assay
  • Requires fewer repeat analyses as a result of a wider reportable range
  • Provides 25-OH-VitD2 and 25-OH-VitD3 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 25-OH-VitD 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-binding assays, including our previous assay: approximately 10% CV at low 25-OH-VitD levels (10 ng/mL)7