SCIENCE & TECHNOLOGY
MASS SPEC WELCOME IN CLINICAL LABS Use of mass spec for CLINICAL DIAGNOSTICS is increasing, but labs face uncertainty about FDA’s plans to regulate tests CELIA HENRY ARNAUD, C&EN WASHINGTON
EVERY YEAR, CLINICAL LABS perform
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.
As new mass spec assays are being develmore than 7 billion tests on blood, urine, oped, many of them are replacing more-esand other samples from patients, according tablished immunoassays. What makes a lab to the American Clinical Laboratory Associdecide that it’s worth the effort to develop, THE STANDARD immunoassay for insuation. Most of those tests involve an immuvalidate, and adopt a new test when they’ve lin-like growth factor-1, which is measured noassay, a diagnostic that uses antibodies to already got one? as a surrogate for human growth hormone, detect molecules of interest. But for a grow“At our laboratory, the decision comes also leaves a lot to be desired. Doctors reing number of tests, mass spectrometry is down to a question of quality,” says Alan L. quest the IGF-1 test to determine whether the method of choice for analyzing samples. Rockwood, scientific director of the mass growth hormone treatment is needed for Labs use the technology for monitoring spectrometry unit at ARUP Laboratories, a children of short stature and to monitor both therapeutic and frequently abused clinical testing service owned by the Univerpituitary function in adults. Analysts have drugs, for performing endocrine tests to assity of Utah. “Is the mass spec method going noticed antibody variability in today’s kits sess various hormonal disorders, and even to be better than the other method?” compared with earlier versions and, thus, for diagnosing infectious diseases. Some of the well-established immunoasvariability in results. “That’s a problem As the use of mass spectrometry says don’t need to be well-documented in the literature,” Clarke OH OH in the clinic grows, however, laboreplaced, says Nigel J. says. ratories face an increasingly unClarke, senior science So Quest developed a mass spec assay for certain regulatory environment. IGF-1 using a high-resolution quadrupole Many immunoassays are run time-of-flight instrument. These instruusing off-the-shelf commercial ments have a broader mass range than the H H kits that have gone through a Food triple quadrupoles that are typically used & Drug Administration approval to detect molecules in clinical process. But almost all mass-spec-based mass spec. The combination of H H assays fall into the category of so-called the extended mass range and HO HO laboratory-developed tests. Such tests the high mass resolution alare cultivated and used in individual lowed Clarke to avoid digesting Vitamin D2 metabolite Vitamin D3 metabolite laboratories without FDA scrutiny. IGF-1 before analysis, a practice CLOSELY RELATED Vitamin D2 and vitamin Instead, they have been and continue to commonly used to obtain fragments small D3 are usually detected as their 25-hydroxy be regulated by the Centers for Medicare & enough for standard mass spec detection. (red) metabolites. Immunoassays can have Medicaid Services. He could instead analyze the intact protein trouble distinguishing the various forms and FDA may soon step in, however. Last Ocand use a well-established extraction procecan be biased toward one or the other. Mass spectrometry can differentiate the parent tober, the agency released a draft framework dure to isolate IGF-1. compounds and the metabolites. for regulatory oversight of lab-developed “We’ve been pushing to make it as simple tests. Some mass spec experts in clinical as possible to prepare the sample,” Clarke labs worry that the proposed regulations says. “A huge amount of the variance and could curtail the continued adoption of director for mass spectrometry and autoerror comes in the sample prep, not in the mass spectrometry by clinical labs. mation at Quest Diagnostics Nichols Inmass spec measurement. If you can make it One way clinical labs use mass spec is stitute, the company’s esoteric diagnostics as simple as possible to get the analyte out to confirm the results of drug screening center, in San Juan Capistrano, Calif. “Let’s and then use the mass spec at the back end, tests. In drug analysis of a urine sample, for face it. We wouldn’t have a diagnostics you tend to get a more accurate result.” instance, a positive result from an immuindustry if we didn’t have immunoassays. Yet another analyte that is increasingly noassay must be substantiated by another There are some really good immunoassays being assayed by mass spectrometry is method. “We use mass spec because of its out there.” vitamin D. Tests for the nutrient typically specificity to reduce false-positive and falseAt the same time, immunoassays have involve measuring the 25-hydroxy metabonegative results,” says Yan Victoria Zhang, problems. “We’ve known how nonspecific lites of vitamins D2 and D3, which are the director of the clinical mass spectrometry antibodies can be,” says Andrew N. Hoofnamain forms circulating in the body. Vitamin and toxicology lab at the University of Rochgle, director of clinical mass spectrometry D is associated with bone health and with ester Medical Center. at the University of Washington. Because some autoimmune diseases. CEN.ACS.ORG
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HEAD-TO-HEAD Immunoassays are the diagnostic of choice in many clinical labs today. But mass spectrometry’s popularity is rising.
Yellow = signal Red = detection antibody Green = capture antibody Blue = analyte
Pro: Cheap and easy Con: Antibodies can cross-react with other analytes Con: Other antibodies can interfere with the test Con: Difficult to be sure what is being detected
Con: Expensive instruments Pro: Fragmentation pattern provides structural information Pro: Better precision and accuracy than immunoassays
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
Q2
Q1
Q3
Signal
Relative intensity
Precursor ion selection
Fragmentation
Fragment selection
Time
NIH
Mass spec In the clinic, the most common analyzer on a mass spec, used to detect molecular fragments, is the triple quadrupole. The first quadrupole (Q1) in the analyzer selects a precursor ion, which then gets fragmented in the collision cell. Specific fragments are selected in the third quadrupole (Q3) for detection.
SHUTTERSTOCK
Immunoassay One antibody captures the analyte and a second labeled antibody binds to the analyte, forming a sandwich. Many types of signaling mechanisms can be used.
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 researchgrade 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 cusCEN.ACS.ORG
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tomers, 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 timeof-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 labdeveloped tests rather than in combination with FDA-approved assay kits. Those labdeveloped 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 regula-
tion 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.” 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
MEDICAL DEVICES 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. Agilent Technologies K6420 Triple Quadrupole MS K6430 Triple Quadrupole MS K6460 Triple Quadrupole MS K6490 Triple Quadrupole MS K6530 Q-TOF MS K6540 Q-TOF MS K6550 Q-TOF MS BioMérieux Vitek MS Bruker MALDI Biotyper CA
Sciex 3200MD QTRAP LC/MS/MS 3200MD Triple Quad LC/ MS/MS 4500MD QTRAP LC/MS/MS 4500MD Triple Quad LC/ MS/MS Shimadzu LCMS-2020 CL LCMS-8030 CL LCMS-8040 CL LCMS-8050 CL
Thermo Fisher Scientific Endura MD Waters Acquity TQD Quattro Micro Quattro Premier Xevo TQ Xevo TQ-S Xevo TQD
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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.” ◾
