CEO SUMMARY: As it developed a home brew mass spec assay for Vitamin 25(OH) D to meet the request of some client physicians, the laboratory at the University of Massachusetts Medical Center quickly recognized several challenges. First, there were fundamental differences in the numbers generated on the same population by the internally-developed LC-MS assay compared to the established immunoassay. Second, physicians were not alert to these differences when results were reported to them.
IN TODAY’S HYPERACTIVE MARKET for Vitamin 25(OH) D testing, the typical clinical laboratory faces at least two challenges as it tries to “get it right” for the physicians and patients it serves.
First, the FDA-cleared immunoassay (IA) that has been in widespread use for almost two decades—and is familiar to most physicians—has an aggressive new competitor: a Vitamin 25(OH) D home brew assay that uses tandem mass spectrometry. Results produced by these different methodologies often do not correlate.
This has caused confusion among clinicians who have long experience with the Vitamin 25(OH) D results produced by the immunoassay method, but may not appreciate how and why the Vitamin D result produced by tandem mass spec can differ from the immunoassay.
Second, labs performing these different methodologies report their results using similar reference ranges. Across the laboratory testing industry, there is neither validation of reference ranges nor an effective effort to educate clinicians about this situation. Not surprisingly, clinicians can often be confused—particularly those clinicians who use multiple labs because of managed care contracts. And, if clinicians are confused, this raises the possibility that their patients may not get a proper diagnosis nor the appropriate treatment for their condition.
In New England, resolving these two issues in Vitamin 25(OH) D testing created significant clinical challenges for the laboratory at the University of Massachusetts Medical Center. This was the subject of a fascinating case study at the recent Executive War College on Laboratory and Pathology Management in New Orleans.
Different Test Methodologies
It was presented by L.V. Rao, Ph.D., who is Director of the Core Laboratory and Immunology at the UMass Laboratory. Rao described how his lab dealt with the requests by some client physicians to provide Vitamin 25(OH) D results by tandem mass spectrometry assay and not the widely-used chemiluminescence immunoassay.
“At UMass, we faced a practical problem with important clinical implications,” stated Rao. “Most all our clinicians are quite comfortable with the long-established chemiluminescence assay (CIA) for Vitamin 25(OH) D. UMass offers the Diasorin CIA. But over the past year, we had several key clients ask us to provide them with a Vitamin 25(OH) D assay by tandem mass spectrometry (LC-MS).
“As we took the steps to develop an in-house tandem mass spec assay to meet the request of these physicians, we faced a number of difficult decisions centered around two primary issues,” he said. “One, the immunoassay method and the mass spec method do not naturally correlate, as each typically produces a unique Vitamin D number from the same sample.
“Two, it is a reality in clinical practice today that the large majority of physicians are familiar with the results and reference ranges for the immunoassay method,” Rao added. “Originally cleared by the FDA in 1993, the immunoassay has been around for almost two decades. Even today, it is the most widely-used method by the largest number of laboratories.
“Therefore, if our lab was to report Vitamin 25(OH) D results by our home brew tandem mass spec method, how would we ensure that the clinician understands that our mass spec result and reference range are often likely to be significantly different numbers than if this same patient’s sample had been tested by the immunoassay method?” he asked.
Here is what makes the experience of the UMass Lab of high interest to other labs evaluating both methodologies for Vitamin 25(OH) testing. As it developed its internally-developed tandem mass spec assay, UMass recognized the potential for clinicians to treat the reported LC-MS results and reference range as equivalent to those of the Vitamin D immunoassay method. If that happened, it might negatively affect patient care. Avoiding this dilemma proved to be a complex challenge for UMass.
“Today, labs have a number of methods available to measure for Vitamin 25(OH) D” said Rao. “These include the classic radioimmunoassays, ELISA 96-well direct assays, chemiluminescence assays, HPLC- UV, and HPLC-MS.
Clinical Studies Of Vitamin D
“In the past two decades, the immunoas- says—specifically radioimmunoassays or chemiluminescence assays—have been used in the vast majority of clinical studies world-wide to define what is normal circulating 25-hydroxy levels,” he observed. “These range from the Harvard cohort studies, such as the Health Professionals Follow-Up Study (HPFS), to the Women’s Health Initiative (WHI) and the National Health and Nutrition Examination Survey (NHANES).
“In recent years, expanded use of tandem mass spec for Vitamin 25(OH) D test- ing has stimulated many publications in laboratory medicine literature,” he continued. “Collectively, these publications show that there are some correlations, some agreement, and some controversy when the Vitamin D immunoassays are compared with the LC-MS assays for Vitamin D.
“Some published findings show substantial agreement between the two meth- ods,” explained Rao, “while other published findings show quite different results generated by these two methods. This is caused by many parameters which are not thoroughly understood.
Explaining The Differences
“Could these differences be due to the assay itself? To variations in performing the assay? Or how the matrix is used?” he asked. “The point here is not enough is understood about how and why the immunoassay method and the mass spec method for Vitamin D generate different answers.”
Having established this context about some important differences between the immunoassay method and the mass spec method for Vitamin 25(OH) D, Rao discussed the steps taken by the UMass laboratory to evaluate the mass spec method and prepare to offer it for clinical use.
“In the past few years, our volume of Vitamin D testing increased dramatically,” recalled Rao. “Go back three or four years ago. At that time, UMass Labs did maybe 300 to 400 Vitamin 25(OH) D tests per month. Right now we perform more than 11,000 tests per month!
Successful Lab Outreach
“Another factor is that our laboratory outreach program is successful at generating new clients,” he continued. “Thus, many of our new client-physicians have recent experience using other labs in their medical practice. In the past year, a few of these physicians emphatically requested that UMass perform Vitamin D by LC-MS method.
“Our initial strategy was to accommodate these relatively few physicians in our outreach program by referring their Vitamin D specimens to Mayo Medical Laboratories,” Rao stated. “Mayo performs the LC-MS method. That seemed like a good solution, until we compared those LC- MS results for our Massachusetts population against the results we generated by our CIA method.
“As you can see from table 1 [sidebar on page 8],” he continued, “there are significant differences in the reference range of our CIA and the Mayo LC-MS assay. There are also significant differences in how each assay method categorizes our Massachusetts population as Vitamin D deficient.
“The data you see draws from January- February 2009,” noted Rao. “First, you will see the differences in the reference ranges of UMass, where above 30 is considered sufficient and Mayo, which uses above 25 as sufficient.
“Next, compare the distribution of the population results. By our CIA method, 68.7% of 16,000 patients were insufficient, under 30,” he said. “But the same population—based on 4,000 tests during those same two months—when tested by Mayo’s LC-MS method and using the cutoff of 25, classifies only 28.2% percent as Vitamin D deficient.
“That shows a significant difference,” explained Rao. “It also presents pathologists and laboratory scientists with a question that cuts to the core of laboratory medicine. If you are a physician, which test methodology for Vitamin D do you use? As this table of actual results demonstrates, depending upon which method is used, the physician can classify somebody as deficient or not deficient. In turn, that drives the decision to treat, or not to treat.
“Next, we looked at what levels of D2 were detected from those 4,000 LC-MS results,” Rao commented. “That is table 2, [see sidebar on page 8]. Almost 75% of the patients had no detectable levels of D2. About 12.5% of patients were at the minimum detectable levels of D2, 4 to 10. The remaining 13% of patients had D2 measurements of between 11 to 162 nanograms per liter. These findings told us that, with our Massachusetts population, around 87% have no or very little amounts of D2 to be detected separately.
Create In-House LS-MS Test
“As we performed these population studies, the volume of LC-MS requests continued growing,” recalled Rao. “That encouraged us to decide, ‘Let’s handle these LC-MS requests with our own in-house assay. We have a toxicology department and we can double up our own assay.’
“As we established our LC-MS assay for Vitamin 25(OH) D, it coordinated very well with the Mayo Medical Lab results,” he continued. “Next we measured about 600 sam- ples simultaneously with both our LC-MS and the immunoassay method. This data is shown in table 3 [see sidebar on page 10].
“The data showed an fairly acceptable correlation (r=0.80), but with significant bias (approximately 40%),” noted Rao. “Informed by this information, we were concerned that the referring physicians who wanted Vitamin 25(OH) D by LC-MS may not understand the fundamental differences produced by this methodology versus immunoassay.”
“We then visited several endocrinologists who had requested us to do their Vitamin D testing by LC-MS,” he said. “We asked them just a few questions, such as: ‘Are you and the majority of physicians in your group aware that there are differences in the methodologies of Vitamin D testing? Did you know about this bias that we see between the two methodologies?’
“Once these endocrinologists saw the comparative data between the two methods of Vitamin D testing, they were surprised,” he added. “They told us that, because different laboratories give them essentially the same reference range—regardless of the methodology—they would not expect to see such a distinctly different bias in the results produced by the IA and LC-MS methods.”
How Docs Use D2
Because endocrinologists typically see patients referred by primary care providers (PCPs), Dr. Rao and his UMass colleagues were intrigued to know how both PCPs and endocrinologists might or might not be using D2 levels. “Our next question to the endocrinologists was “Do you get a patient referral because the PCP sees a D2 level reported as ‘undetectable’ and, possibly because of confusion about the significance of this number, then decides to refer the patient on to the specialist?’
“Their answer was not what we expected,” Rao went on. “The endocrinolo- gists told us that the vast majority of questions they get about low Vitamin D levels actually come from patients who have direct access to their laboratory test reports. These patients see the ‘undetectable’ D2 result on their test report and ask their doctor ‘Am I vitamin D deficient?’
“Informed by these insights, we asked if there would be specific advantage in their clinical practice to know the D2 level compared to the total Vitamin 25(OH) D level reported by immunoassay method,” recalled Rao. “Every doctor told us that, for patient screening purposes, there is no specific advantage to having the D2 and D3 subtotals reported along with a total Vitamin D. However, when a physician is monitoring a patient taking D2 therapy, then it would be appropriate to know the D2 level of that patient.
“This interaction with our client physicians helped us educate those physicians about the issues in using the two methods for Vitamin 25(OH) D testing,” observed Rao. “However, in the larger game, it leaves us with an ongoing issue.
“We are a laboratory now offering clinicians the option of Vitamin 25(OH) D by the long-established, FDA-cleared chemiluminescence assay and by our home brew LC-MS assay,” he noted. “We know and understand the differences in the results and the reference ranges we provide for each method.
“But a large number of physicians in practice today remain unaware that these important differences exist between the two methodologies,” continued Rao. “As a result, the potential exists for these clinicians to make an inaccurate diagnosis and thereby possibly fail to provide the most appropriate treatment to the patient. As a laboratory, we would like to eliminate that imprecision, but there is no help from the national literature.
“What is missing to help clinicians— and the laboratories which serve them—are specific guidelines and agreements published in the broader clinical community regarding the appropriate clinical intervals of Vitamin D,” offered Rao. “The literature provides no consensus on a specific level of 25-hydroxy indicator of Vitamin D deficiency.
“Further, the majority of the many studies published over the years used radioimmunoassays and chemiluminescent assays,” he observed. “Recommended optimum levels have come from these studies. But this situation does not provide effective guidance to labs and physicians for use of the LC-MS method in determining Vitamin D levels.
“This is the gap, so to speak, in the current clinical practice concerning use of LC- MS in Vitamin D testing,” stated Rao. “We laboratorians recognize the precision of the LC-MS method, which requires a highly- trained and dedicated technician. It’s very accurate and interference-free compared to immunoassays.
“However, as demonstrated in our laboratory’s experience providing physicians with Vitamin D results generated by both methods, for the LC-MS assay to have clinical relevance, I believe LC-MS results must agree with the immunoassay,” offered Rao. “Accomplishing such a correlation has been the unsolved challenge for our laboratory.
Explaining Bias Factor
“For example, from our 600-patient population, we did establish a bias factor for our home brew LC-MS assay relative to the Liaison CIA that we’ve used for years,” he explained. “Next, we went to several client physicians and asked them, ‘Should we establish a bias factor and represent both the LC-MS value and the immunoassay equivalence to you on the test report?’
“These physicians initially liked this idea,” noted Rao. “So, we offered both LC- MS measured values and calculated immunoassay equivalent values in the patient report. This created confusion among some physicians as to what number for a Vitamin D result was appropriate upon which to base their clinical actions. In response to this, we removed the calculated value from the patient test reports.”
Complications For Clinicians
This experience of the laboratory at the University of Massachusetts Medical Center demonstrates how the widening use of LC-MS assays for Vitamin 25(OH) D testing can bring complications to the clinical environment. For clinicians, unaware of the fact that the LC-MS methodology typically produces different (and often higher) numbers than the long-accepted immunoassay methodology, there is the potential for them to inappropriately evaluate the patient. In turn, that might mean the patient gets the wrong treatment for their true condition.
This same situation often causes physicians to question the credibility of the laboratory which reported the Vitamin D result. These doctors will contact the reporting laboratory and question the accuracy of the Vitamin D results it reported. This is often the case when a physician is using multiple labs because of managed care contracts.
Rao believes some resolution to this situation may come from a new development. “On July 14, the National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health’s Office of Dietary Supplements (NIH-ODS,) announced the development of a new reference sample for Vitamin D,” stated Rao. “It is called NIST Standard Reference Material (SRM) 972–Vitamin D in Human Serum.
“This SRM will provide stable, well-defined levels of the analytes of interest,” he explained. “It will serve as a reproducible point of comparison, of results across differ- ent methods and within the lab over time.
“NIH-ODS also announced an ODS-funded NIST quality assurance program for analysis of Vitamin D metabolites in human serum,” concluded Rao. “NIST chemists will compile data and provide the laboratory with confidential feedback about its performance These are positive first steps towards standardization of Vitamin D testing.”
As the experience of the laboratory at the University of Massachusetts Medical Center demonstrates, the widening use of LC-MS assays for Vitamin 25(OH) D testing has brought complications to the clinical environment. For clinicians, unaware of the fact that the LC-MS methodology typically produces different numbers than the long-accepted immunoassay methodology, there is the potential for them to inappropriately evaluate the patient. In turn, that might mean the patient gets the wrong treatment for their true condition.