Q&A column

Q. A nephrology patient who has been treated with vitamin D₂ for several years contacted our laboratory to find out why their 25-hydroxyvitamin D level of 60 ng/mL is now considered elevated when before it was within the normal range. How can we explain this?

A.Vitamin D is a fat-soluble vitamin that regulates calcium and phosphate metabolism and supports bone homeostasis. It is also thought to have a wide range of extraskeletal effects via the nuclear vitamin D receptor, which is expressed throughout the body.

As a preprohormone, vitamin D exists as D₂ (ergocalciferol) and D₃ (cholecalciferol). Ergocalciferol is plant based and can be found in fortified foods, dietary supplements, and prescription form. Cholecalciferol is present in animal-based products and is synthesized in the skin following exposure to ultraviolet B light. Both must be activated through hydroxylation in the liver to generate 25-hydroxyvitamin D (25[OH]D) and in the kidneys to yield 1,25-dihydroxyvitamin D (1,25[OH]₂D).

The body stores a significant amount of vitamin D as 25(OH)D, which is the preferred analyte to assess for vitamin D sufficiency (≥20 ng/mL), insufficiency (12 to <20 ng/mL), and deficiency (<12 ng/mL).^1,2 Measuring 25(OH)D is appropriate in populations at high risk of vitamin D deficiency, including people with malabsorption, hyperparathyroidism, osteoporosis, obesity, and limited exposure to sunlight.

Although vitamin D deficiency is a risk factor for a variety of diseases, randomized trials of vitamin D supplementation for primary or secondary prevention of falls, fracture, cancer, cardiovascular disease (including stroke and myocardial infarction), depression, and autoimmune disease have proved unsuccessful.^3,4 Screening of vitamin D levels in asymptomatic adults or during pregnancy is not recommended.^5,6

The reader’s question raises two concerns that need to be addressed. First, the patient asked why their previously normal value is now considered high. Unfortunately, there is no consensus regarding the upper limit of the reference range for 25(OH)D. Some laboratories align the upper limit of the normal range with studies that demonstrate toxicity when serum 25(OH)D levels exceed approximately 80 ng/mL. However, levels above approximately 50 ng/mL are not typically observed in healthy people.

A comprehensive review by the National Academy of Medicine found that almost all people are vitamin D sufficient when serum 25(OH)D levels are greater than 20 ng/mL and that values greater than 50 ng/mL may indicate over supplementation or toxicity.² Many laboratories have aligned their reference interval to a range of 20–50 ng/mL. Therefore, it is possible that the patient’s laboratory changed its reference range to align with the National Academy of Medicine’s recommendations.

However, recommended cutoffs may not be relevant for all laboratories since interlaboratory comparisons have demonstrated that measured serum 25(OH)D levels vary by methodology, assay manufacturer, and instrument, with poor agreement among immunoassays. Immunoassay-measured serum 25(OH)D levels are often inaccurate when compared to levels from liquid chromatography-tandem mass spectrometry-based reference methods.^7,8

The second concern is that 25(OH)D₂ does not reliably cross-react with immunoassay detection reagents, leading to under-recovery of 25(OH)D₂, which contributes to interassay variability. While LC-MS/MS-based 25(OH)D assays can accurately quantify 25(OH)D₂ and are generally more accurate than immunoassays, the high levels of the C-3 epimer of 25(OH)D₃ observed in neonates and infants may be insufficiently resolved on chromatography, leading to overestimation of 25(OH)D₃ levels.^7,8

Therefore, when testing for vitamin D is warranted, it is critical to understand the limitations of the individual assay and of the reference interval being employed to meaningfully interpret test results and limit clinician and patient confusion.

1. Giustina A, Adler RA, Binkley N, et al. Controversies in vitamin D: summary statement from an international conference. J Clin Endocrinol Metab. 2019;104(2):234–240.
2. Ross AC. The 2011 report on dietary reference intakes for calcium and vitamin D. Public Health Nutr. 2011;14(5):938–939.
3. Guirguis-Blake JM, Michael YL, Perdue LA, Coppola EL, Beil TL. Interventions to prevent falls in older adults: updated evidence report and systematic review for the US Preventive Services Task Force. JAMA. 2018;319(16):1705–1716.
4. Manson JE, Cook NR, Lee IM, et al. Vitamin D supplements and prevention of cancer and cardiovascular disease. N Engl J Med. 2019;380(1):33–44.
5. Holick MF, Binkley NC, Bischoff-Ferrari HA, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2011;96(7):1911–1930.
6. American College of Obstetricians and Gynecologists. Vitamin D: screening and supplementation during pregnancy. Committee opinion No. 495. Obstet Gynecol. 2011;118(1):197–198.
7. Erdman P, Palmer-Toy DE, Horowitz G, Hoofnagle A. Accuracy-based vitamin D survey: six years of quality improvement guided by proficiency testing. Arch Pathol Lab Med. 2019;143(12):1531–1538.
8. Wise SA, Phinney KW, Tai SS, et al. Baseline assessment of 25-hydroxyvitamin D assay performance: a Vitamin D Standardization Program (VDSP) interlaboratory comparison study. J AOAC Int. 2017;100(5):1244–1252.

Andy Hoofnagle, MD, PhD
Professor, Laboratory Medicine
Head, Division of Clinical Chemistry
Department of Laboratory Medicine and Pathology
University of Washington
Seattle, Wash.
Chair, CAP Accuracy-Based Programs Committee

Rebecca Treger, MD, PhD
Assistant Professor
Department of Laboratory Medicine and Pathology
University of Washington
Seattle, Wash.
Member, CAP Diagnostic Immunology and Flow Cytometry Committee