Vitamin D is an important building block for bone heath, as it is required for maintaining overall calcium homeostasis (1). Endocrinologists agree that it is challenging to obtain adequate vitamin D intake through diet and limited sun exposure, requiring supplementation and fortification of foods; even if they don’t agree on the optimal dosage of said supplementation.

At one point, large reference laboratories were running an astonishing 5 million vitamin D tests per year, or more. Despite no recommendation for generalized screening for hypovitaminosis D, this testing volume was largely due the emphasis on the ‘potential health benefits of vitamin D’ in the lay media. Since then, randomized controlled trails have not consistently concluded on the benefits of vitamin D supplementation beyond supporting overall bone health.

There are several instances where testing 25-hydroxyvitamin D (25(OH)D) to monitor supplementation, for sufficiency or risk of toxicity, and the assessment of vitamin D stores is essential to quality patient care (2):

  • Patients with clinical symptoms or presentation of poor bone health
    (rickets, osteopenia/porosis, osteomalacia, or history of non-traumatic fractures)
  • Increased metabolism and catabolism of 25(OH)D
    (primary hyperparathyroidism or hypercalcemia of malignancy)
  • Malabsorption syndromes
    (inflammatory bowel disease, Crohn’s disease, bariatric surgery)
  • Decreased production of 25(OH)D
    (liver disease or hepatic failure)
  • Altered production of 1,25(OH2)D
    (kidney disease, granulomatous disease)
  • Patients taking medications known to increase the risk of poor musculoskeletal health
    (antiseizure medications, glucocorticoids or HIV/AIDS medications)

Introduction of traceable liquid chromatography-mass spectrometry (LC-MS/MS) reference measurement procedures (RMPs), incorporating standard reference materials (SRMs), has significantly increased the accuracy in 25(OH)D determination. However, even with assay standardization and decreased inter-laboratory imprecision under the CDC Vitamin D Standardization Program (VDSP), it appears many participants are not meeting the performance criteria for the majority of certification samples evaluated (3). Here, participants receive certification based on average performance (mean bias ±5.0% and mean imprecision of <10% CV), but the number of individual samples passing these criteria appears to be as low as 25% of the 40 survey samples for 2018 (median:55%, range:25-90%, n=20 participants).

We have also recently demonstrated the inaccuracies of 25(OH)D immunoassays in real patient samples (4). This is particularly true in samples containing appreciable (>20 ng/mL) 25(OH)D2 where a significant proportional bias was observed when compared to a traceable LC-MS/MS reference method. Considering the volume of 25(OH)D testing, the incidence of samples containing >20 ng/mL 25(OH)D2 represents more than 3,000 samples annually (8% of the population studied). Even though vitamin D3 is now available in the same high-dose (50,000 IU) weekly or monthly prescription form previously only available as vitamin D2, we have observed sustained vitamin D2 supplementation in a various healthcare networks across the US.

Of the SRMs available through NIST and CDC, only one contains an adequate concentration of 25(OH)D2 to ensure the accuracy of these methods. The remaining SRMs have concentrations of 25(OH)D2 ranging from 0 to 2.6 ng/mL and offer little to support standardization efforts. Additional SRMs, such as those through the University of Ghent Labquality Reference Serum Panel, for 25-Hydroxyvitamn D may help support further standardization efforts.

Proficiency testing surveys have historically been limited in challenging the accuracy of methods, especially the ability to recover 25(OH)D2. Unless external quality assurance surveys include a sufficient number of proficiency testing samples from patients supplemented with 25(OH)D2, assay performance, including the realization of manufacturers’ stated recoveries, will not be adequately assessed.

The clinical consequences of inaccurate 25(OH)D determination may seem relatively benign. However, under-recovery may lead to unnecessary diagnostic testing and clinical work-up for malabsorption or result in prolonged high-dose supplementation that could potentially lead to downstream clinical sequelae of hypervitaminosis D.


  1. Tolan NV. Vitamin D. In: Rifai N, ed. New England Journal of Medicine Knowledge+ and American Association for Clinical Chemistry Learning Lab Adaptive Learning Program, 2017. (Accessed 9/5/2018).
  2. Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2011;96:1911-30.
  3. Centers for Disease Control and Prevention (CDC) Vitamin D Standardization-Certification Program (VDSCP): Total 25-Hydroxyvitamin D Certified Procedures. Hormone and Vitamin D Standardization Programs. Accessed September 2018.
  4. Tolan NV, Yoon EJ, Brady AR, Horowitz GL. Price of High-Throughput 25-Hydroxyvitamin D Immunoassays: Frequency of Inaccurate Results. The Journal of Applied Laboratory Medicine 2017. JALM.2017.024323; DOI: 10.1373/JALM.2017.024323