ORLANDO, Florida — Inappropriate orders for the 1,25-dihydroxy vitamin D test have been increasing in recent years, according to the authors of a paper being presented at the College of American Pathologists (CAP) 2025 meeting.

Two tests for vitamin D predominate in the US, Rita Khoury, MD, laboratory director of Aculabs in East Brunswick, New Jersey, noted: the 25-hydroxy (25-OH) test, the most common test, which measures vitamin D stores and deficiencies, and the 1,25-dihydroxy test, which should be used only if a patient has hypercalcemia, granulomatous diseases, chronic kidney failure, or hyperparathyroidism. 

According to Choosing Wisely recommendations from the American Academy of Family Physicians, “Serum levels of 1,25-dihyroxy vitamin D have little or no relationship to vitamin D stores but rather are regulated primarily by parathyroid hormone levels, which in turn are regulated by calcium and/or vitamin D. In vitamin D deficiency, 1,25-dihydroxyvitamin D levels go up, not down.”

“However, in the last 8-10 years, we have noticed an increase in the 1,25-dihydroxy vitamin D test,” Khoury told Medscape Medical News. The numbers stood out because most labs cannot handle the test in-house and must send it out to specialists, adding expense and delaying turnaround times. She estimated the cost of the 1,25-dihydroxy test is “probably double” that of the 25-OH test, although the prices vary by laboratory.  

“The majority of the 1,25-dihydroxy vitamin D (orders) are not necessary,” Khoury added.

Previous literature has recommended the benchmark ordering ratio of 1,25-dihydroxy vitamin D tests to 25-OH tests at 1:50. Khoury’s team analyzed data on 268,412 samples for 25-OH vitamin D and 5502 samples for 1,25-dihydroxy vitamin D between January 2011 and August 2025 and found the ratio appears to be narrowing. It stood at 1:47 in 2011 but peaked at 1:240 in 2015. In 2021, the ratio narrowed to 1:32 as interest in the potential immune benefits of vitamin D soared in the early years of the COVID-19 epidemic. This year, the ratio was at 1:37 when the researchers checked the data.

“The 25-OH vitamin D test volume has not gone down,” Khoury said. Her team found a rise from 10,722 in 2011 to more than 26,000 a year in 2025. But the volume of the 1,25-dihydroxy test has substantially outpaced that growth, with orders rising more than fourfold, from 230 per year to 1059 per year in that period.

Electronic Search May Fuel the Problem

She says one of the problems is the automation of ordering the tests in the electronic health record and the way the tests appear in the dropdown menu when a clinician searches for vitamin D. Online ordering exploded from 12.1% in 2011 to over 98% by 2023, she noted.  

“With an automated system, when you put in vitamin D, the first one that’s going to come to you in the dropdown, numerically, is 1,25,” she explained. At her company, when 1,25-dihydroxy is ordered, a pop-up message asks clinicians to confirm they truly want to request the test. 

Pathologists are frustrated with the inappropriate ordering because “we don’t see the patient; we see the test that was ordered,” Khoury said. Ordering the wrong test can lead to misleading normal results in patients with true vitamin D deficiency.

She said education needs to extend not only to physicians but to nurses, medical assistants, and anyone involved in the ordering process. 

“It’s a struggle that many labs have,” Gabrielle Winston-McPherson, PhD, assistant professor in the Department of Pathology & Laboratory Medicine at University of Wisconsin-Madison, told Medscape Medical News.

Message Warns of Mix-ups

Winston-McPherson said confusion about when to use each test has persisted for “at least 15 years” and a warning was added to Wisconsin’s test directory to help clinicians avoid mistakes. Now when their clinicians order a test for 1,25-dihydroxy vitamin D, a message is attached saying: “Do not order for vitamin D status except for renal patients. For vitamin D status, the correct test to order is vitamin D 25-hydroxy.”

But just adding that information might not be enough. “They may not see all that information when they are initially searching the order,” she said. “What we learn when we use search engines is that the most popular or most appropriate one should come up first. But that’s not necessarily how the search functions in our EMRs [electronic medical records] work.”

Winston-McPherson said it is also “a strong possibility” some clinicians might not remember there are two different tests for vitamin D or know when to use one or the other. Having more firm guidance at the point of the order may help, she said, such as an alert that would interrupt the order and explain the appropriate use. The only downside to that is the potential for increasing alert fatigue, she said.

“Problems like this, while they might seem minor, are really significant,” she said.

Khoury and Winston-McPherson reported having no relevant financial conflicts of interest. 

Marcia Frellick is a Chicago-based healthcare journalist and a regular contributor to Medscape Medical News.