Laboratory Testing for Vitamin D Deficiency
Order a serum 25-hydroxyvitamin D [25(OH)D] level using an assay that measures both 25(OH)D2 and 25(OH)D3 forms. This is the single most important test for diagnosing vitamin D deficiency, as 25(OH)D is the primary circulating form and the most accurate biomarker of vitamin D status 1.
Primary Diagnostic Test
- Serum 25-hydroxyvitamin D [25(OH)D] is the only laboratory test needed to diagnose vitamin D deficiency in most clinical scenarios 1, 2, 3.
- The assay must measure both 25(OH)D2 (ergocalciferol metabolite) and 25(OH)D3 (cholecalciferol metabolite) to capture total vitamin D status, as patients may be taking either form of supplementation 1.
- Deficiency is defined as 25(OH)D <20 ng/mL, insufficiency as 20-30 ng/mL, and optimal levels as ≥30 ng/mL 2, 3, 4.
Tests That Should NOT Be Ordered
- Do not order serum 1,25-dihydroxyvitamin D [1,25(OH)2D] for diagnosing vitamin D deficiency, as this active form provides no information about vitamin D status and is often normal or even elevated due to secondary hyperparathyroidism in deficient patients 3.
- Measuring 1,25(OH)2D is a common pitfall that leads to missed diagnoses, as clinicians may incorrectly assume normal vitamin D status when seeing normal or high 1,25(OH)2D levels 3.
Additional Laboratory Considerations
- Serum calcium and parathyroid hormone (PTH) may be obtained in patients with severe deficiency (<10-12 ng/mL) to assess for secondary hyperparathyroidism and hypocalcemia, though these are not required for diagnosis 5, 6.
- Serum phosphate and alkaline phosphatase can help identify osteomalacia in severe cases but are not necessary for routine diagnosis 6.
- Avoid using gel tubes for blood collection when possible, as recent data suggest potential interference with 25(OH)D measurement by LC-MS/MS methods 1.
Important Testing Caveats
- Population-wide screening is not recommended by major organizations including the USPSTF, which concluded there is insufficient evidence to support screening asymptomatic adults 1, 2.
- Testing should be reserved for high-risk individuals: those with osteoporosis, malabsorption syndromes, chronic kidney disease, dark skin pigmentation, limited sun exposure, obesity, elderly/institutionalized persons, and those on medications affecting vitamin D metabolism 2, 4.
- Laboratory variability exists between different 25(OH)D assays and even between laboratories using the same method, so using assays calibrated to international standards is critical 1, 2.
- Inflammation can falsely lower 25(OH)D levels when C-reactive protein (CRP) >40 mg/L, complicating interpretation in acutely ill patients 5.
When to Recheck Levels
- After initiating treatment, recheck 25(OH)D levels at 3 months minimum to allow levels to plateau and accurately reflect response to supplementation 1, 5.
- If using intermittent dosing regimens (weekly or monthly), measure levels just prior to the next scheduled dose 1.
Special Populations
- For dark-skinned, veiled, elderly, or institutionalized individuals with limited sun exposure, empiric supplementation with 800 IU/day without baseline testing is reasonable and cost-effective 1, 2.
- In chronic kidney disease patients (GFR 20-60 mL/min/1.73m²), measure 25(OH)D as these patients are at particularly high risk due to reduced synthesis, dietary restrictions, and urinary losses 5.