CEO SUMMARY: Those labs performing Vitamin 25(OH) D testing by mass spectrometry face an interesting challenge. For more than two decades, physicians, patients, and a majority of credible clinical studies have accepted RIA and IA Vitamin 25(OH) D results as a familiar standard. That is why, to avoid confusing physicians, some labs using the mass spec method correlate those mass spec results to the IA method and report those correlated results with the immunoassay reference ranges.
DO ANY RESEARCH INTO VITAMIN D TESTING and you will quickly find the name of Bruce W. Hollis, Ph.D. Since 1981, Hollis has been a national figure in Vitamin D research and an expert on Vitamin D testing.
Hollis is Professor of Pediatrics, Biochemistry, and Molecular Biology; and Director of Pediatric Nutritional Sciences at the Medical University of South Carolina in Charleston, South Carolina. As one of the nation’s leading authorities on Vitamin D, Hollis has been closely observing the evolution of Vitamin D testing in this country.
“My interest in Vitamin D testing goes back 25 years,” stated Hollis. “I’ve developed radioimmunoassay (RIA) tests for Vitamin D. In conjunction with DiaSorin, Inc., we developed the first 25-hydroxy- vitamin D (25 OH Vitamin D) test to receive FDA clearance back in the early 1990s. This test has been widely used to determine the normal level of Vitamin D.”
In fact, over the past two decades, virtually all major studies of Vitamin D have utilized the RIA or Laision immunoassay tests developed by Diasorin and Hollis. Because of that fact, clinicians are quite familiar with the reference ranges for these RIA and IA assays.
Hollis has watched other assays for Vitamin D arrive in the marketplace. “Back in 2000, Nichols Institute Diagnostics (NID), a division of Quest Diagnostics Incorporated, introduced the Nichols Advantage 25-Hydroxy Vitamin D Assay,” he noted. “This automated test was not RIA. Instead, it used a human binding protein as a binding agent.
Protein Binding Agents
“In fact, protein binding agents for Vitamin D testing had been used for years and they never worked satisfactorily in direct assays,” continued Hollis. “The NID Advantage 25D test was no exception. It was a disaster that created quite a bit of chaos when it was pulled from the market a few years ago.
“One consequence of the failed NID assay was that many laboratory scientists did not trust any type of binding assay,” he said. “It didn’t matter if it was an antibody, a protein, or similar binding agent. Several laboratories decided to solve this problem by using mass spectrometry to measure Vitamin D, a technology which has the potential to be very precise.
“These labs jumped right in and developed their own Vitamin 25(OH) D assay using liquid chromatography–tandem mass spectrometry (LC-MS/MS),” observed Hollis. “Several of these labs then offered this LC-MS/MS Vitamin D test to clinicians without calibrating their results with the DiaSorin 25(OH) D assay.
False Positive Situation
“That created a problem,” explained Hollis, “The mass spec method tends to produce a number which can be 40% or more higher than what the DiaSorin test would generate on the same patient sample. Thus, when those labs report their uncalibrated mass spec results against the DiaSorin range, physicians may get mass spec Vitamin D results (uncorrelated to the DiaSorin assay) which look normal against the DiaSorin range, but actually represent a false negative test report.”
One of those laboratories that used LC- MS/MS to establish an in-house assay for Vitamin 25(OH) D was Quest Diagnostics. “When Quest began using the mass spec method for Vitamin D testing several years ago, it reported its mass spec results against the DiaSorin normal range, but it had not calibrated those results against the DiaSorin assay,” stated Hollis.
Payers Changed Labs
“Meanwhile, during this same period, Laboratory Corporation of America was using the DiaSorin method for Vitamin 25(OH) D testing,” he commented. “What happened was insurance carriers would switch back and forth between Quest and LabCorp in their provider networks. That meant physicians would use Quest for a long time, until the health insurer switched to LabCorp as the exclusive lab provider.
“When that happened, Vitamin 25(OH) D results reported to physicians by LabCorp came back much lower than what the physicians had seen when these patients had been earlier tested by Quest” he noted. “So physicians called LabCorp saying ‘you don’t know what you’re doing! You don’t know how to run a proper test. We’ve been using Quest and they’ve told us that mass spec is the gold standard.’
“Also at this time, John J. Cannell, M.D., a psychiatrist in California who founded the Vitamin D Council, began receiving a steady stream of questions and complaints from patients and physicians,” Hollis stated. “They asked why there was such an unsettling difference in the Vitamin D results reported by Quest and LabCorp for the same patient. Dr. Cannell was one of the first medical professionals to recognize this problem and call public attention to it.”
Cannell is interviewed on pages 6-9. He has been outspoken in describing the negative patient consequences when laboratories report mass spec Vitamin 25(OH) D results using the DiaSorin normal range without also calibrating the mass spec test result against the DiaSorin assay.
Mayo Calibrated To Diasorin
“To my knowledge, during this time, Quest had never calibrated its Vitamin 25(OH) D mass spec test to the DiaSorin assay,” Hollis said. “That’s in contrast to Mayo Clinic, From day one, it set up its Vitamin 25(OH) D mass spec assay to be matched and calibrated to the DiaSorin test. Ravinder Singh, Ph.D., at the Mayo laboratory, frequently swapped samples with DiaSorin for calibration reasons to see how samples match up. Singh’s mass spec Vitamin D test seems to correlate quite well with the Diasorin assay.
“Some labs offering Vitamin D tests by mass spec did it the right way and correlated results to match the widely accepted ranges familiar to clinicians. If a laboratory wants to report its Vitamin D mass spec result using the higher numbers that can result—as much as 40% higher—then it should raise the range it reports to physician. But there are still some labs using mass spec that have yet to do that.”
Hollis recognizes that Mass spec is a detection system with the capability to be more specific than a UV detection system. But he also cautions that a wide disparity in Vitamin 25(OH) D mass spec results can be seen in the clinical market. “As I travel and give lectures, I often hear it said that mass spec is a ‘gold standard’ for Vitamin 25(OH) D testing. But if that is true, then why is there such variability in results among different labs?” he asked.
“For example, DiaSorin sent samples to seven labs running mass spec and got seven different results!” commented Hollis, who is a consultant to DiaSorin. “So I ask you, which one of these seven results would you consider the gold standard? This is frustrating for everyone concerned about quality and consistency in diagnostic testing. After all, mass spec is like any diagnostic technology. If you put garbage in, you’ll get garbage back.”
Integrity Of Test Results
Hollis notes that mass spectrometry is like other diagnostic technologies. Every required step in specimen collection, transport, preparation, and analysis must be done correctly for the test result to be accurate and reproducible. “Variability in the integrity of the test result can be affected by several factors, not the least is sudden, unexpected surges in the volume of specimens to be tested,” said Hollis.
“In such circumstances, with a rapid run up in the volume of specimens, to maintain turnaround time, the lab staff may fail to properly prep all the samples,” observed Hollis. “There is often a lack of consistency in how different people on different shifts set up and run the test, particularly if the lab is hiring new people to process the extra work.
“Second, these problems are particularly aggravated when the increased vol- ume of specimens overwhelms the technical skills of the lab,” he added. “If the Ph.D. and key technologists lack experience and competency in this type of testing, that can affect the quality of results reported by the lab.
“There have been reports that indicate that Vitamin D testing at Quest ramped up rapidly from 50,000 tests per month to possibly as many as 500,000 tests per month in about 24 months,” noted Hollis. “That would be overwhelming for any laboratory. It places great stress on quality control and the ability to generate high quality, reproducible test results.”
Cannell and Mercola First To Alert Docs, Patients
PHYSICIANS JOHN CANNELL, M.D., AND JOSEPH MERCOLA, D.O., were among the first to recognize that differences in how the nation’s two largest laboratories reported Vitamin D results had the potential to confuse doctors and patients alike.
Both are opinionated, outspoken individuals who maintain active Web sites which are visited by tens of thousands of people each month. As these two doctors became aware of how variation in the way results were reported by some labs using mass spec for Vitamin 25(OH) D testing was causing confusion with physicians and patients alike, both doctors alerted the public to this situation. Both doctors also publicly recommended that physicians and patients send specimens to both LabCorp and Quest Diagnostics, then compare the results to understand the problem.
“I applaud John Cannell and Joe Mercola for pressing this issue because it was being ignored,” noted Bruce Hollis, Ph.D., Professor of Pediatrics, Biochemistry, and Molecular Biology at the Medical University of South Carolina. “They accurately described the problem and provided useful guidance on how physicians and patients should take steps to ensure that, when testing for Vitamin 25(OH) D, they understand the meaning of the different test results they often see from different labs.”