Every year, clinical labs perform more than 7 billion tests on blood, urine, and other samples from patients, according to the American Clinical Laboratory Association. Most of those tests involve an immunoassay, a diagnostic that uses antibodies to detect molecules of interest. But for a growing number of tests, mass spectrometry is the method of choice for analyzing samples. Labs use the technology for monitoring both therapeutic and frequently abused drugs, for performing endocrine tests to assess various hormonal disorders, and even for diagnosing infectious diseases.
As the use of mass spectrometry in the clinic grows, however, laboratories face an increasingly uncertain regulatory environment. Many immunoassays are run using off-the-shelf commercial kits that have gone through a Food & Drug Administration approval process. But almost all mass-spec-based assays fall into the category of so-called laboratory-developed tests. Such tests are cultivated and used in individual laboratories without FDA scrutiny. Instead, they have been and continue to be regulated by the Centers for Medicare & Medicaid Services.
FDA may soon step in, however. Last October, the agency released a draft framework for regulatory oversight of lab-developed tests. Some mass spec experts in clinical labs worry that the proposed regulations could curtail the continued adoption of mass spectrometry by clinical labs.
One way clinical labs use mass spec is to confirm the results of drug screening tests. In drug analysis of a urine sample, for instance, a positive result from an immunoassay must be substantiated by another method. “We use mass spec because of its specificity to reduce false-positive and false-negative results,” says Yan Victoria Zhang, director of the clinical mass spectrometry and toxicology lab at the University of Rochester Medical Center.
As new mass spec assays are being developed, many of them are replacing more-established immunoassays. What makes a lab decide that it’s worth the effort to develop, validate, and adopt a new test when they’ve already got one?
“At our laboratory, the decision comes down to a question of quality,” says Alan L. Rockwood, scientific director of the mass spectrometry unit at ARUP Laboratories, a clinical testing service owned by the University of Utah. “Is the mass spec method going to be better than the other method?”
Vitamin D2 and vitamin D3 are usually detected as their 25-hydroxy (red) metabolites. Immunoassays can have trouble distinguishing the various forms and can be biased toward one or the other. Mass spectrometry can differentiate the parent compounds and the metabolites.
Some of the well-established immunoassays don’t need to be replaced, says Nigel J. Clarke, senior science director for mass spectrometry and automation at Quest Diagnostics Nichols Institute, the company’s esoteric diagnostics center, in San Juan Capistrano, Calif. “Let’s face it. We wouldn’t have a diagnostics industry if we didn’t have immunoassays. There are some really good immunoassays out there.”
At the same time, immunoassays have problems. “We’ve known how nonspecific antibodies can be,” says Andrew N. Hoofnagle, director of clinical mass spectrometry at the University of Washington. Because antibodies sometimes bind to the wrong analytes, “immunoassays are fraught with errors that mass spec can actually solve.”
Testing for testosterone is a prime example. High levels of testosterone found in healthy men can easily be measured with immunoassays. But measuring the naturally low levels of the hormone found in women, children, and hypogonadal men requires the extra sensitivity of mass spec. That’s because other hormones and metabolites present in samples can compete with testosterone to bind to the immunoassay’s antibodies, interfering with the results.
The standard immunoassay for insulin-like growth factor-1, which is measured as a surrogate for human growth hormone, also leaves a lot to be desired. Doctors request the IGF-1 test to determine whether growth hormone treatment is needed for children of short stature and to monitor pituitary function in adults. Analysts have noticed antibody variability in today’s kits compared with earlier versions and, thus, variability in results. “That’s a problem well-documented in the literature,” Clarke says.
So Quest developed a mass spec assay for IGF-1 using a high-resolution quadrupole time-of-flight instrument. These instruments have a broader mass range than the triple quadrupoles that are typically used to detect molecules in clinical mass spec. The combination of the extended mass range and the high mass resolution allowed Clarke to avoid digesting IGF-1 before analysis, a practice commonly used to obtain fragments small enough for standard mass spec detection. He could instead analyze the intact protein and use a well-established extraction procedure to isolate IGF-1.
“We’ve been pushing to make it as simple as possible to prepare the sample,” Clarke says. “A huge amount of the variance and error comes in the sample prep, not in the mass spec measurement. If you can make it as simple as possible to get the analyte out and then use the mass spec at the back end, you tend to get a more accurate result.”
Yet another analyte that is increasingly being assayed by mass spectrometry is vitamin D. Tests for the nutrient typically involve measuring the 25-hydroxy metabolites of vitamins D2 and D3, which are the main forms circulating in the body. Vitamin D is associated with bone health and with some autoimmune diseases.
Immunoassays for vitamin D have a tough time distinguishing between vitamins D2 and D3 and their metabolites because they are so structurally similar. The diagnostics instead report a total vitamin D value. And some immunoassays are biased toward one type of vitamin D over the other, which can lead to errors in the overall number.
The molecular weight information clinicians obtain via mass spec makes it possible to distinguish among the two types of vitamin D and their metabolites. “For vitamin D, a robust LC/MS/MS method provides superior quality over the immunoassay in terms of accuracy, precision, and reproducibility,” says Julia C. Drees, scientific director of chemistry at Kaiser Permanente Regional Laboratory in Richmond, Calif. Using this mass spec method, she says, “we do 2,500 vitamin D tests per day.”
Mass-spec-based vitamin D assays have not been without issues. In 2008, Quest needed to recall the results from a large number of mass-spec-based vitamin D assays because of calibration problems at some of its labs. The company hasn’t experienced quality issues with the assay since then, according to company spokeswoman Wendy H. Bost. Quest has recently started offering an improved immunoassay for detecting vitamin D, but mass spec remains the preferred method, Bost says.
Most labs have developed and run their clinical mass spec assays on research-grade instruments. But as the use of mass spectrometry in clinical diagnostic labs has grown, instrument companies have responded by listing some of their mass spectrometers with FDA as Class I medical devices. These instruments are functionally equivalent to their “research-use only” counterparts that have been manufactured in facilities not registered with FDA.
Class I medical devices are general-purpose instruments that don’t have specified intended uses. Instead, clinical labs can use those instruments in their own lab-developed tests.
“Our customers had started using mass spectrometry for clinical purposes because they had requirements to be able to do testing in a way that is sensitive, in a way that is cost-effective, in a way that gives them fast turnaround times that weren’t being met by other technologies,” says Tamara Smith, global clinical market manager at Sciex. “To be compliant in marketing to this set of customers, we needed to have our instruments be medical devices.”
The registration process ensures that companies meet certain quality standards in the manufacturing of instruments. One way companies choose to do that is by complying with the ISO 13485 Quality Management Standard for Medical Devices, which focuses on risk management and design control of instruments. In addition, each company must set up postmarket surveillance, including adverse event reporting.
Some companies have developed assay kits that can be combined with mass spectrometers. Such kits lift the burden from individual labs to develop and validate assays and could provide a way for labs to easily comply with anticipated FDA requirements. For example, in the U.S., Waters offers a test kit for measuring the immunosuppressant drug tacrolimus. In certain other regions, the company markets test kits for additional immunosuppressant drugs. Sciex has developed kits for immunosuppressants, newborn screening, and vitamin D measurements that are approved for use in Europe but not in the U.S.
Bruker has taken the extra step of obtaining FDA approval for its MALDI Biotyper CA system as a Class II medical device, which means that it is tied to a particular assay. The system is based on Bruker’s microflex time-of-flight instrument and is approved for use in microbiology labs for infectious agent identification. It uses an algorithm to match the proteomic pattern of a sample against those in a well-characterized library.
So far, clinical labs have been using mass spectrometers almost exclusively for lab-developed tests rather than in combination with FDA-approved assay kits. Those lab-developed tests have so far been regulated by the Centers for Medicare & Medicaid Services under the Clinical Laboratory Improvement Amendments of 1988 (CLIA). But FDA is in the process of establishing a framework for regulating the tests as medical devices. FDA claims that it has always had the authority to regulate lab-developed tests but has until now chosen to exercise “enforcement discretion.” The draft framework was published last October, and the comment period closed in February.
“We’ve been working through the numerous comments that were given,” says Alberto Gutierrez, director of FDA’s Office of In Vitro Diagnostics & Radiological Health. If the revisions are extensive, FDA may need to go through another round of comments. So it’s difficult to predict when the agency might release final guidance.
In the meantime, the lab community is opposed to FDA regulating lab-developed tests. “FDA has good intentions in trying to make sure that laboratories are not doing poor-quality work,” says the University of Washington’s Hoofnagle. But FDA regulation is not the answer. Instead, he argues, CLIA should be modernized to require that scientists provide enough detail on a laboratory-developed test to ensure that the test has been properly validated.
“Validation is very different from regulation,” Hoofnagle says. “Validation is already being done.”
Hoofnagle considers regulation to be “the set of rules that surround the practice of medicine,” some of which can be frivolous. “We are an incredibly regulated industry already,” he says. “For many labs, some of the changes that FDA is proposing are changes in paperwork that will not benefit the patient.”
In contrast, validation is “a sincere attempt to demonstrate the robustness of a system,” Hoofnagle says. “It is a series of experiments and tests that makes us comfortable that we did a good job designing and developing an assay.”
Hoping to ensure their mass spectrometers continue to find use as clinical diagnostics, instrument makers have listed some of their mass spectrometers with the Food & Drug Administration as medical devices. All are Class I general-purpose medical devices, except for the Bruker MALDI Biotyper CA and BioMérieux Vitek MS, both of which are Class II medical devices intended for identifying infectious agents.
K6420 Triple Quadrupole MS
K6430 Triple Quadrupole MS
K6460 Triple Quadrupole MS
K6490 Triple Quadrupole MS
6530 Q-TOF MS
6540 Q-TOF MS
6550 Q-TOF MS
MALDI Biotyper CA
3200MD QTRAP LC/MS/MS
3200MD Triple Quad LC/MS/MS
4500MD QTRAP LC/MS/MS
4500MD Triple Quad LC/MS/MS
Thermo Fisher Scientific
Richard C. Friedberg, the medical director of Baystate Reference Laboratories in Springfield, Mass., and president-elect of the College of American Pathologists, an organization that accredits diagnostic labs, likewise agrees that FDA regulation is the wrong approach but that there are problems with many assay validations.
“We’ve got to know to what extent measurements are accurate, reliable, and reproducible,” Friedberg says. “Too many places have done wimpy validations. Maybe that’s where we need to have ideas—how you should properly do a validation.”
Clarke points out that labs are already regulated under CLIA, and some states, especially New York, have even stricter requirements. Before a test can be used in New York, the state department of health must approve the validation and standard procedures. In addition, these various regulating authorities require labs to undergo proficiency testing to show that their personnel can accurately run analyses.
“People don’t realize how much oversight we’re already under,” Clarke says. “We’re not saying we don’t want to be regulated. It’s just a question of whether you’re going to gain very much above and beyond where we already are.”
Even if FDA ends up regulating lab-developed tests, it plans to let labs continue offering tests while the approval process is ongoing. “If a test is approved, they would have been able to stay on the market through the whole thing,” Gutierrez says. “If we do not approve it, at that point they would have to stop.”
In addition to doubts about FDA’s regulatory authority over lab-developed tests, labs are worried that the process will be expensive. For example, in a comment submitted to FDA, Robert Schmidt of ARUP estimated that it would cost more than $300 million for the company to comply with FDA’s regulatory framework as currently stated. (Only a fraction of ARUP’s lab-developed tests use mass spectrometry.)
FDA’s Gutierrez believes that estimates such as this one are overinflated. “If you’ve already developed a test, if you’ve already validated your test—and one hopes you did clinical validation of your test because one hopes you’re offering a test that is clinically valid—then you should have sufficient data already to show that the test is actually clinically valid.”
Industry response runs the gamut from “people who are completely shrugging it off” to “people who are absolutely scared,” says Brian Rappold, scientific director at Essential Testing, a company that specializes in tests for pain medication monitoring.
But if the uncertainties surrounding FDA’s intentions can be resolved, people see a bright future for mass spectrometry in clinical labs.
“I believe we really are in the golden age of mass spectrometry,” Rappold says. And to make sure it continues, mass spec users in clinical labs need to be open about what works, what doesn’t, and how to fix problems, he says. “We don’t want mass spec to languish in just a research setting. We all believe in the power of mass spectrometry being part of the standard clinical tool kit. It’s got a lot of work to get there.”