Diabetes Goals: the GooD anD baD newsNew research published in Diabetes Care indicates that while important strides were taken between 1988–1994 and 2007–2010 among diabetics in achieving target HbA1c, blood pres-sure (BP), and low-density lipoprotein cholesterol (LDL-C) goals, disparities in certain subgroups suggest an “urgent need” for additional treatments and care.

The findings came from a nation-ally representative sample of 4,926 participants in the National Health and Nutrition Examination Surveys from 1988–1994, 1999–2002, 2003–2006, and 2007–2010. Participants, 20 years or older, self-reported a previous diagnosis of diabetes and completed household surveys and physical exami-nations. American Diabetes Associa-tion (ADA)-recommended levels of HbA1c, BP, and LDL-C, and current use of statins were the outcomes measured.

The study’s good news is that in the most recent time period, 18.8% of participants achieved all three goals—HbA1c <7.0%, BP <130/80 mmHg, and LDL-C <100 mg/dL—compared with just 1.7% in 1988–1994. The researchers attributed these gains to the advent of new and improved therapeu-tic agents and more evidence about the benefits of risk factor control.

At the same time, however, the authors found some troubling trends. Most notably, about 30% of younger adults, Hispanics, and those with dia-betes for at least 20 years had HbA1c levels ≥8.0%. In addition, approxi-mately 40% of those taking insulin had HbA1c values ≥8.0%.

The authors hypothesized that poor diabetic control in these groups might stem from several factors. Some people might not be able to achieve ADA-recommended targets due to the severity of their disease or complica-tions. In addition, they might lack self-management skills, resources to adhere to demanding self-care regimens, or individualized care. Furthermore, cur-rent thinking recognizes that not every person should be expected to reach the targets.

The authors concluded that manag-ing diabetes “remains very complex and challenging, requiring access to a skilled team of clinicians and diabetes educators and imposing major bur-dens on families and health systems.”

ClinicalLaboratoryNews

n e w s b r i e fThe auThoriTaTive

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may 2013volume 39, number 5

w w w . a a c c . o r g

The Rise of Mobile Health DevicesThe Next Big Advance for Diagnostics? By Bill Malone

The cellular phone, which recently marked its 40-year anniversary, has evolved from a 2 ½-pound, brick-like device to a palm-sized, 4-ounce mobile computer more powerful than most early desktop machines. Today, the cellular networks that power smartphones

have become so pervasive and so powerful that mobile Internet us-age is ready to surpass desktop usage. With smartphones and other mobile devices connecting wirelessly to almost anything digital, an explosion of mobile-capable devices is rapidly emerging as a billion-dollar industry.

A study published recently in Clinical Chemistry gives a glimpse into this future—powerful, miniaturized instruments paired with cellular networks and cloud computing that work even in remote parts of the world (Clin Chem 2013;59:629–40). In the study, re-searchers demonstrated that their rugged, credit card-sized micro-fluidic chip, called the mChip, could perform lab-quality HIV tests in Rwanda. In this case mobile means more than just small: the mChip device automatically synced results with patients’ remote electronic health records (EHR) over cellular networks—all at lower cost and in less time than bench-top equipment laboratorians use every day in modern, sophisticated labs.

The marriage of diagnostics and mobile connectivity means more than just a new stream of gadgets, ac-cording to cardiologist, geneticist, and mobile health expert Eric Topol, MD. “I actually think this is the next

A Call for Better Estradiol MeasurementHow Quickly Can Improvements Be Made?By Genna Rollins

research over the decades has demonstrated that the sex steroid hormones, primarily estradiol and testosterone, not only exert control over the development and maintenance of reproductive capacity but also have a guiding hand on a good many other biological processes. The evolving understanding of these latter functions has placed new measurement demands on both estradiol and testosterone, particularly at low levels that reflect non-reproductive physiology. In the case of

testosterone, a concerted effort during the past 5 years by a broad public-private consortium has made great strides in achieving more robust and standardized measurements. Now, an Endocrine Society panel has turned

its sights on estradiol measurement, calling for a universally recog-nized traceable standard, age- sex- and biologically-specific reference ranges, and more.

“We have the same problem with estradiol as we’ve had with tes-tosterone, which is that the current assays, particularly direct immu-noassays, are inadequate for many of the purposes we need estrogen measurements for. That includes in men, post-menopausal women, and in adolescents and children,” said the panel’s chair, William Ros-ner, MD, a professor of medicine at the Columbia University College of Physicians and Surgeons in New York City. “This is a very am-bitious agenda, and we don’t think it’s going to happen tomorrow.

See mobile DX, continued on page 3

See estradiol, continued on page 6

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snapshoTAchieving Targets in Diabetes*

Source: Diabetes Care 2013; doi: 10.2337/dc12-2258

* Percentages based on 4,926 participants

1988–1994 2007–2010

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big wave of where the diagnostics field will go,” Topol said. “It’s basically an out-growth of the remarkable progress that’s been made in microfluidics. For example, if you connect lab-on-a-chip hardware to a smartphone, that smartphone can do a lot of the processing, display, and archiving of the data. Already the likes of thyroid func-tion tests, liver tests, and electrolytes are in the pipeline. This is definitely happening.” Topol is professor of genomics and the di-rector of the Scripps Translational Science Institute in La Jolla, Calif. He previously chaired the department of cardiovascular medicine at Cleveland Clinic, and last year was voted the Number One Most Influen-tial Physician Executive in Healthcare by Modern Healthcare.

Tiny Instruments, Big ImpactThe lab-on-a-chip concept has fueled pre-dictions about mobile diagnostics for years. But few of these devices have proven suc-cessful in practice. According to mChip designer Samuel Sia, PhD, lead author of the study in Clinical Chemistry, researchers have to look beyond pure analytical perfor-mance. “You can have the best performing technology in the world, but if it requires too many steps by the user or complicat-ed equipment, it will never be used in the field,” he said. “One really needs to think about how to integrate every component from the beginning: fluid handling, signal amplification and detection, data commu-nication, as well as user experience—all of this has to work together seamlessly.” Sia is an associate professor of biomedical en-gineering at Columbia University in New York City and co-founder of Claros Diag-nostics, now called OPKO Diagnostics.

To be sure, the mChip is a workhorse. The device captures all the essential func-tions of an ELISA in an elegant, injection-molded translucent plastic card, with re-agents tucked away inside a nearly invisible lattice of precision channels (See Photo, p. 5). A compact reader initiates the test and displays results with the push of a sin-gle button. In the Clinical Chemistry study, the mChip demonstrated 100% sensitiv-ity and specificity for HIV using 1 µL of whole blood from a finger prick, outper-forming traditional lateral-flow rapid tests and matching bench top ELISA perfor-mance in under 15 minutes. Importantly, the mChip also correctly identified weakly

positive samples, the Achilles heel of many rapid HIV tests based on traditional tech-nology.

However, Sia and his colleagues didn’t stop there. They incorporated two wire-less communications technologies into the compact mChip reader: a satellite transceiver for global coverage and an-other for local cellphone towers. This way, results automatically became part of pa-tients’ EHRs, along with a date and loca-tion stamp, no matter where or when the test was performed. Such a mobile uplink to EHRs enables healthcare workers to monitor outbreaks of disease, strategically allocate medications and other resources, begin treatment faster in remote settings, and reduce the errors inherent in manual

transcription of results, according to the researchers.

Although the compact reader for the mChip displays results clearly to the user, Sia’s team had a broader clinical need in mind when they integrated cellular con-nectivity. “We wanted to build a real prod-uct, not just show proof of concept, and if you want to build a product that people will use, it’s important to consider what the clinical need actually is,” Sia explained. “In this case, our aim is to improve care for pregnant women in remote settings in the developing world. When a woman comes in for testing, it’s vital that the health-care worker know the patient’s history, so whatever testing they perform needs to be linked to both past and future patient re-cords. That’s why we designed the device to automatically sync with the patient’s elec-tronic record.”

Engineering for Cost and SimplicityCurrently available rapid tests, such as lat-eral flow-based HIV screening kits now available over the counter, often rely on pushing the limits of traditional methods in order to muster the requisite sensitivity and specificity. The emerging microfluidic methods exemplified by the mChip have to move in the opposite direction: they start with high performance, but their intricate design can threaten usability and reliability. For microfluidics to become part of useful, mobile devices, engineers face an uphill climb, Sia said.

“Our approach is to take a platform that is intrinsically high performing, and then try to reduce its complexity so that it can be affordable and usable in the field,” Sia said. “Affordability and usabil-ity are not just an afterthought. You can’t

See Mobile DX, continued on page 4

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Mobile Extends Reach of EHRsMobile DX, continued from page 1

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single-mindedly pursue performance, and expect only later on to consider making all the steps automated. That kind of thinking doesn’t lead to a wholly integrated device that works well for the user.” Sia believes his research team can get the cost down to about $1 per test for the mChip, compared to $1.80–$6.20 for a standard bench top ELISA. The total cost of the mChip system is about $1,000 versus $20,000.

Topol, who recently published a book, The Creative Destruction of Medicine: How the Digital Revolution Will Create Better Healthcare, has kept a close eye on mobile health companies. Last year, he co-authored an opinion article in Clinical Chemistry enumerating a handful of cutting-edge technologies he believes signal the advent of “unplugged laboratory medicine” (Clin Chem 2012;58:1644–7). “By using a cus-tomizable array of sensors, reagents, and substrates, microfluidics devices serve as miniature factories that ‘shrink the plumb-ing’ typically found in traditional laborato-ries into a palm-size device. The advantages are substantial: speed, portability, and capa-bility to perform a wide array of biochemi-cal testing,” Topol wrote. “Integration with smartphones will ultimately help emerging lab-on-a-chip devices bridge the existing gap between bioengineering prototypes and commercially viable clinical tools. By providing mobile access to computing power and Internet connectivity, smart-phones also offer sensors, cameras, and high-resolution displays that can be used to visualize data and enable telemedicine ap-plications.”

However, for those mobile health tech-nologies that do prove themselves effec-tive, cost remains a big hurdle, Topol told CLN. “One of the biggest problems that we

always confront at the intersection of tech-nology and medicine is: will it reduce costs? There is a long history of new, innovative technology coming in and actually raising costs,” he said. “That has to be resolved.”

It’s hard not to compare lab-on-a-chip devices to the computing hardware that’s become commonplace in modern life. Computers that once filled entire rooms have rapidly shrunk in size and increased in power to the point that computing power is mostly invisible, encapsulated in tiny mi-croprocessors hiding inside smartphones, televisions, and toys. In some ways, the lab-on-a-chip arena has followed a narra-tive similar to the computing evolution in Silicon Valley, taking each step of perform-ing an assay and making it progressively smaller and faster.

According to Sia, lab-on-a-chip micro-fluidics is certain to evolve, but lab-quality test results on mass-market, consumer-oriented devices will take time. “I think the time scale is still an open question, in terms of when we’ll get to that point where people can diagnose a range of conditions on their smartphone,” he said. “I think that point will happen. In fact it’s probably inevitable that it will happen. But if we were to push the analogy, I think where we are now with diagnostics is probably comparable to the early 1980s of computing. Most diagnostic tests are still run in centralized labs, and the question is, can we miniaturize these tests to the point where the everyday consumer can really use them. If people think putting together an iPhone is hard, putting togeth-er one of these diagnostic mobile health devices is at least an order of magnitude harder, because you have to integrate so many things together—not only software and hardware, but the chemistry and biol-ogy that are notoriously fickle.”

Liberating DataMicrofluidic technology will no doubt continue to fascinate and inspire engineers, laboratorians, and diagnostics companies. However, mobile health is about more than just new diagnostic technology. Familiar instruments can take on new power when they incorporate mobile connectivity. Take the humble blood glucose meter used by millions of people with diabetes. Last year, Bethesda, Md.-based Telcare, Inc. launched the first-of-its-kind cellular-enabled meter that automatically uploads results to an online portal—no smartphone required. With the data in the cloud, patients can provide their caregivers secured access to customized graphs and reports to make sense of the patients’ myriad of daily test results.

With nearly 30,000 units sold, Telcare has branded its wireless meter as both a pa-tient engagement device and a population health management tool. Patients can view serial reports about their glucose readings online or on companion smartphone apps (See Screen Image, p. 5). They also can re-ceive customized education and other mes-sages based on their test results, right on their meter.

According to David Bjork, Telcare’s president and COO, liberating the data from blood glucose meters opens up a host of new uses for test results that otherwise would remain hidden or forgotten on con-ventional meters. “The whole notion is about making use of this data to drive pa-tient engagement, which in turn motivates patients to be more self-aware and more educated,” Bjork said. “We can also observe this data and then do something with the observation in order to assist that person. We can even alert a healthcare professional when a patient is struggling and not mak-ing wise decisions.”

This can make a big difference for pa-tients living with diabetes who many times

Microfluidics Still Evolving RapidlyMobile dX, continued from page 3

Mobile Health Market Poised for growthFDA Vows Not to Suppress Innovation in New Regulations

According to experts, the mobile health industry is still in the early stages of a growth curve. A report published in March from the Berlin-based mobile market research firm research2guidance estimates that 500 million smartphone users worldwide will be using a healthcare app by 2015. The report pre-dicts that in 5 years, the so-called mHealth market will achieve “mass market status,” with 50% of the estimated 3.4 billion smartphone users having downloaded mHealth applications. In the 2013–2017 period, the firm forecasts market revenue growth of 61%, reaching $26 billion. To put this number in per-spective, that’s nearly a third of what market research-ers expect the in vitro diagnostics market to reach in the same timeframe, $80 billion.

As recently as 2011, the market for health-oriented mobile apps began to change, according to the re-port. Whereas previously the business model revolved around a pay-per-download model for smartphone apps, the ascending model has relied on services, companion devices, and in-app purchases. Now, free applications serve as platforms for mobile health services and hardware. For example, the iHealth blood pressure app that charts daily readings is free, but the accompanying blood pressure cuff and iPhone dock costs $72.99 in retail stores.

Taking the idea a step further, imaging and other devices that depend on mobile computing power have already hit the market, such as AliveCor’s Food

and Drug Administration (FDA)-cleared electrocardio-gram with a sensor that snaps to the back of an iPhone.

Pending regulations for mHealth from FDA will also play a role in driving growth. Ironically, the lack of regulation has actually hampered more rapid growth in health-related mobile devices, according to research2guidance co-founder Ralf-Gordon Jahns. “The uncertainty around regulation has stalled a lot of companies in the mHealth area,” he said. “You can’t build a solid business case around an mHealth device when you don’t know what the FDA is going to require or which apps could be regulated as medical devices.”

FDA released a draft guidance in 2011 cover-ing how it would regulate health-related apps, but the agency left a lot of gray area that only served to increase anxiety for mHealth app and device devel-opers. In a March 21, 2013 hearing convened by the House Energy and Commerce Committee, Christy Foreman, director of the FDA’s device evaluation division, sought to assure Congress that the agency would not stifle innovation, and promised to release a final version of the long-awaited guidance in October. She emphasized that only apps that sought to per-form functions of bona fide medical devices would come under scrutiny from the agency.

Once FDA acts, it could prove to be a turning point for the health-related sector of the mushrooming mobile market, Jahns said.

CliniCal laboratory news May 2013 5

feel abandoned after an initial diagnosis to manage their own care, Bjork said. “Those patients who are very ill do receive attention from the healthcare system, but that’s only about ten percent of those living with the disease,” he said. “The majority of people with diabetes are basically on their own—until they require expensive interventions. Our healthcare system doesn’t do a good job right now of managing the entire popu-lation of people living with diabetes.”

With a new cache of patient data in hand, Telcare is also building bridges to providers’ EHRs, making test results that once died on patients’ meters at home part of the same records available in hospitals or doctors’ offices. While the company re-mains focused on diabetes, how successful

this integration becomes could bode well for other mobile testing platforms.

Physicians, especially endocrinologists, are beginning to use the Telcare portal. But so far the greatest interest has been from payers and other risk-bearing orga-nizations, such as self-insured employers, commercial insurance companies, and ac-countable care organizations (ACO), Bjork noted. Telcare is working to analyze the data points from thousands of wireless me-ter results and pinpoint individuals most at risk. Payers see the potential here to identify which patients need extra help before more costly interventions are required, saving the system money in the long run.

“In some respects, the integration has been more intense on the payer side. These payer organizations that bear financial risk for populations find the information we’re collecting very valuable,” Bjork said. “An or-ganization that bears financial risk is much more apt to stand in the front of the line and say, I want to deploy this platform because it can change my risk profile. If you can ob-serve patient behavior, then you can identify risks in the population and start proactively supporting people that are otherwise man-aging their conditions by themselves.”

ACOs, value-based contracts among insurers and providers, and similar reim-bursement models that emphasize pay-ing for value versus fee-for-service drive adoption, according to Bjork. “The minute that this type of contract is in place, our platform becomes very attractive, because

physicians now have an incentive to pay attention to patients between office visits. With our platform, they can monitor that population of patients for whom they now carry a financial risk.”

A Consumer-Driven MarketOf course, most mobile health apps and devices don’t have a long history inside the healthcare system like a blood glucose meter. Companies seeking to break into mobile health from the outside will have to focus on appealing directly to consumers.

A recent mobile health market report from Berlin-based firm research2guidance predicts the market will grow to $26 bil-lion within 5 years (See Box, p. 4). How-ever, most of this growth will likely not come from Food and Drug Administra-tion (FDA)-cleared products covered by insurance such as Telcare’s blood glucose meters. Rather, companies will depend on consumers dipping into their own pockets, according to Ralf-Gordon Jahns, cofounder of research2guidance. “A con-sumer-driven market means that an app or companion device will have to be so at-tractive and so useful to patients that they are willing to pay for it themselves,” he said. “That means that you have to have a more consumerist design, with a product that

is very easy to use and understand. For at least the next few years, companies simply can’t rely on most payers in the healthcare system to be quick to adopt mobile health solutions.”

Topol emphasized that he believes many healthcare professionals underesti-mate consumers. He expects mobile health to become a positive feedback loop, in which increasingly empowered consum-ers demand more and more. “I feel that a lot of people don’t respect the capability, desire, and motivation of patient consum-ers,” Topol said. “My experience has been that when patients have access to their data, it’s such a decided advantage. It switches people who would be the least likely to be data-driven to become data-driven.”

In addition, now that consumers have an appetite for mobile health technology, social media have the ability to drive adop-tion much faster than in the past, Topol added. “We have a special situation today where new products can go viral very rap-idly with social networking, so that a con-sumer has a much higher level of power to bring in other consumers than ever before. If this gets legs among some key parties, adoption of mobile health apps and de-vices could happen a lot faster compared to other products in the past.” CLN

Samuel Sia, PhD, holds his mChip device, a microfluidic card that can perform up to 10 lab-quality ELISA assays in less than 15 minutes.

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A companion iPhone app neatly graphs data from the wireless Telcare blood glucose meter. The meter automatically uploads test results for analysis via an online portal by physicians or other caregivers authorized by patients.

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But at the end of the day, how can we as physicians practice medicine without trust-ing the numbers we’re getting from these assays?”

New Uses Outpace MethodsPanelist Patrick Sluss, PhD, recalled that the uses of and demands on estradiol as-says have changed considerably since the immunoassays to measure this hormone were first developed. “Back in the 1970s, we used estradiol testing primarily in younger women for fertility management where measuring relatively high levels was the target,” said Sluss. “Since that time our picture of both estradiol and testosterone as being gender-specific hormones has changed. However, whether you’re look-ing at estradiol in men or testosterone in women, you need very low levels of detec-tion. Whereas the assays were designed to measure testosterone in men and estrogen in women, which, in both cases, are at high levels.” Sluss is director of special chemistry at Massachusetts General Hospital and as-sociate professor of pathology at Harvard Medical School in Boston.

Levels of estradiol in women of child-bearing age usually are >60 pg/mL, de-pending on the day of the menstrual cycle. Women undergoing induction ovulation for infertility treatment might have con-siderably higher levels, 250–2,000 pg/mL. In contrast, levels in post-menopausal women, men, and pre-pubertal children and adolescents typically are <20 pg/mL, and even lower, <5 pg/mL, in elderly men and women. The lowest levels, <1 pg/mL, probably can be found in women with breast cancer receiving aromatase inhibitor therapy to suppress estradiol levels. To meet the measurement needs of these disparate populations, the panel suggested that estra-diol methods must be capable of yielding results over an impressive range, by a factor of 10,000 (J Clin Endocrinol Metab 2013; 98:1353–60).

That tall marching order poses chal-lenges for just about all methods available to measure estradiol. The lower limit of quantitation of first-generation direct im-munoassays is 30–100 pg/mL, which, even with a coefficient of variation of 20%, is adequate for use in premenopausal women and those undergoing fertility treatments. Liquid chromatography tandem mass spectrometry (LC-MS/MS) and second-generation direct immunoassays have lower limits of detection, about 10 pg/mL, but are not in all cases robust enough to capture the normal distribution of values for populations known to have low estra-diol concentrations. High-performance (HP) LC-MS/MS potentially has adequate sensitivity and specificity for low estradiol concentrations, but still is not without po-tential analytical pitfalls. Similarly, the gold standard method for measuring estradiol, isotope dilution gas chromatography-MS, is too complex and time-consuming for routine clinical use (See Figure, above).

Rosner explained that each successive assay method has made it easier to meet the growing need for estradiol testing, but all also have their own challenges. “The early radioimmunoassays involved ex-tracting the biological fluid, followed by a

chromatography step, and then the assay itself. They had good sensitivity and speci-ficity at that time,” he said. “To get more efficient, direct immunoassays were devel-oped which didn’t require extraction and chromatography steps. That led to the abil-ity to automate, which drove the cost down considerably. However, the price of that au-tomation was very often inaccuracy. Direct immunoassays work fine in some cases, but not very well in others. Estradiol and testos-terone are examples of that.”

Arguably one of the most significant challenges for direct immunoassays is that antibodies in these tests cross-react with multiple forms of the hormone. According to the panel, estradiol circulates in blood as more than 100 conjugated and unconjugat-ed metabolites. Furthermore, patients may have circulating estrogens from exogenous sources such as conjugated equine estro-gens and nutritional supplements. Some of these forms circulate in high enough concentrations that even small cross- reactivities can cause “profoundly deranged results,” the panel wrote.

The Limits of Mass Spectrometry Even as the authors cited problems with direct immunoassays, they were quick to point out that MS methods have short-comings too. “Although these technologies have a number of theoretical advantages over immunoassays, they too may show considerable variability, as has been shown for other analytes such as testosterone,” the panel wrote. “As is true for all methods, measurements based on mass spectrom-etry depend upon the accuracy of calibra-tion, freedom from interference, and ap-propriateness of any analytical corrections. Thus, it is important to realize that it is not solely the method per se that determines validity and utility.”

Panelist Hubert Vesper, PhD, com-mented on the pros and cons of MS-based methods for measuring estradiol. “Mass spectrometry does have some advantages in terms of specificity of the measure-ment and in some cases also sensitivity of the measurement. Mass spectrometry can provide very accurate measurements if it’s performed correctly, but it’s a very complex technology and some of the problems we see in it are the same ones we see with im-munoassays,” he said. “This means we need to make sure that mass spectrometry as-says, as well as immunoassays, need to be calibrated properly.” Vesper is director of clinical standardization programs and chief of the protein biomarker laboratory in the Centers for Disease Control and Preven-tion’s (CDC) Division of Laboratory Sci-ences.

Rosner elaborated that while MS has come along over the years in terms of sim-pler instrumentation and faster through-put, it also is not a panacea for measuring estradiol, a point echoed by panel mem-ber Margaret Wierman, MD, who has had her own difficulties with this method. “As anybody knows who’s tried to develop one, steroid assays for mass spectrometry are very difficult. I spent two years trying to do it here and we failed,” she said. “We have to be a little careful about the new kid on the block, which is expensive and labor inten-sive. We have to prove that it’s better before

we jump on it.” Wierman is a professor of medicine and neuroscience at the Univer-sity of Colorado School of Medicine and chief of endocrinology at the Denver VA Medical Center.

Fine Variances Make a Big DifferenceWierman went on to explain that regardless of method, a host of analytical issues—in-tra- and inter-assay variability, inadequate sensitivity and specificity, and interfer-ences—combine to yield inaccurate and inconsistent estradiol results, complicating the diagnostic process. “The current plat-form assays can’t distinguish between 10 and 60 pg/mL. That can lead you in two directions. Very low numbers cause you to think something’s wrong so you do an extra set of tests to evaluate for what you think might be wrong. On the other hand, you don’t realize something is wrong be-cause the assay you’re using doesn’t have sensitivity at the low levels or sensitivity or specificity at the high levels,” she said. “I just can’t tell you how many wasted evaluations I’ve done over the last four-to-five years be-cause of available assays. In endocrinology practice, we rely on hormone tests, and if we can’t depend on the results to help di-agnose normal and pathologic states, we’re sort of stuck.”

She elaborated that for many endocrine disorders, fine differences in reported estra-diol concentrations substantially influence clinicians’ thoughts about differential diag-noses. For instance, a woman menstruating irregularly could be under stress or experi-encing early menopause. In a workup for this patient, Wierman would evaluate both follicle stimulating hormone (FSH) and estradiol results. Small concentration dis-parities in either hormone could lead her to

draw different conclusions, with profound consequences for the patient. “If the FSH is high, but the estradiol is low, that sug-gests early menopause. But if this is a young woman, this is heart-breaking news, so I wouldn’t want to tell her that unless I was certain. I have to decide: do I tell her, do I not, and how much more do I evaluate be-fore I know for sure?” she said.

Minor estradiol testing inaccuracies can spell trouble for managing diseases, too. Wierman cited as an example managing disorders of puberty, where the transition to sexual maturity starts too early or too late. “You need to have the right numbers to target, not too low, not too high,” she said.

Lack of analytical accuracy not only im-pacts current clinical care but also is hold-ing back a better understanding of emerg-ing science around estradiol’s role beyond female reproduction and sexual maturity. “One of the conundrums we see—and this is not 100 percent correct but is true in a general sense—is that women don’t have heart attacks until they’re menopausal. The question is, is that because their estradiol disappears?” said Rosner. “On the other hand, there are some papers to indicate that men with higher estradiol are at greater risk for heart attacks. That literature is quite confusing, but it’s pretty clear that estra-diol is involved with the heart and arterial health. There’s a lot to be learned.”

The panel cited a number of areas where there is incomplete or conflicting data, in-cluding estradiol’s role in men not only in normal physiology and pathophysiology, but also in bone health, muscle strength, hypogonadism, and hormone-dependent cancers.

Given all the challenges with measuring estradiol, the panel called first and fore-

Method Accuracy Tops List of ChallengesEstradiol, from page 1

Analytical Sensitivity of Estradiol Assays

Lower limits of quantitation for first-generation direct immunoassays are sufficient, even with a 20% coefficient of variation, to measure the range of values in pre-menopausal women and those undergoing fertil-ity treatment. Other populations, including post-menopausal women, men, and prepubertal children, typically have concentrations at or below the limits of quantitation for other methods.

Legend: CV, coefficient of variation; LC-MS/MS, liquid chromatography tandem mass spectrometry; RIA, radioimmunoassay; MDD, minimal detectible dose

Source: Rosner W, Hankinson SE, Sluss PM, et al. Challenges to the measurement of estradiol: An endocrine society position statement. J Clin Endocrinol Metab 2013;98: 1353–60. Copyright 2013, The Endocrine Society. Used with permission.

CliniCal laboratory news May 2013 7

most for a universally recognized estradiol standard to which all other measurements can be traced. In addition, the group rec-ommended development of age- sex- and biologically-specific reference ranges. The committee emphasized the need for wider recognition that estradiol measurements using whole serum without prior extrac-tion are unlikely to be reliable, especially in populations known to have low values. Finally, the authors called for new methods to measure estradiol in the 0.2–2 pg/mL range in routine clinical specimens.

The PATH ForwardIf the panel’s analysis of the current state of estradiol testing seemed downbeat, their forecast for its future was equally optimis-tic. This in no small part is due to strides made recently in standardizing testosterone measurements. Much of the heavy lifting for this has been accomplished by CDC’s hormone standardization project (HoST), which seeks to ensure accurate and com-parable results across assay methods, labs, and time. In the case of testosterone, HoST within just a few years has developed a ref-erence method for HPLC-MS/MS calibrat-ed to a reference material, NMI-M914, with another reference material, SRM 971, used as a trueness control. This process assured metrological traceability. The agency also is working with both manufacturers and labs to calibrate assays, and it collaborates with proficiency testing programs, including the College of American Pathologists and New York State.

Beyond CDC’s efforts is the Partner-ship for the Accurate Testing of Hormones (PATH), a consortium of at least 14 orga-nizations, including AACC, with a goal of advancing standardization and harmoni-zation of hormone assays. PATH has taken several crucial steps toward achieving tes-tosterone standardization, including sup-porting a male reference interval project and planned female and pediatric projects, undertaking several educational initiatives, and urging medical journal editors to en-courage and eventually require the use of accuracy-based testosterone assays in any studies they publish.

As a long-standing, tireless advocate of improved hormone testing, this has been music to Rosner’s ears. “I can’t tell you how pleased I am at how well HoST and PATH efforts have been accepted and how they have changed the whole landscape of testosterone testing,” he said. He and other members of the panel now hope to pass these same milestones on the road to stan-dardize estradiol testing.

AACC also is banking on PATH’s suc-cess with testosterone measurements to guide the way for estradiol, according to Gary Myers, PhD, vice president of pro-grams and policy. “The success of PATH again emphasizes the need for standard-ization of clinical laboratory results to improve patient care. This is really im-portant because it will take a combined partnership effort between the laboratory community, the diagnostic manufactur-ers, and clinical groups to implement standardization improvement in clini-cal laboratory results. With the experi-ence gained in standardizing testoster-one measurement, the effort to improve estradiol measurement is a natural next step.” Myers added that PATH’s success is important to AACC because it directly

supports the Association’s strategic initia-tive around harmonization, the Interna-tional Consortium for Harmonization of Clinical Laboratory Results.

Already, CDC has taken several impor-tant steps, according to Vesper. The agency has developed a LC-MS/MS reference method for estradiol, which it is using to assign target values to single donor serum materials that labs can use to assess the cali-bration and accuracy of their assays. CDC also has other efforts underway.

The Here and Now for Labs Before these initiatives take root, laborato-rians can do much to improve the use and understanding of estradiol measurements. Wierman and Sluss urged labs as a starter to communicate clearly to physicians when they change their assay methods. “Our

methods are driven by lots of things, in-cluding economics. With multi-analyte platforms we very often are making meth-od decisions that move from one vendor to another, and with something like estradiol where it’s very method-dependent it cre-ates a lot of use problems,” explained Sluss. “The most important thing for laborato-rians is to develop good communications with endocrinologists and to work together on how those tests are being ordered, used, and interpreted.”

Wierman also implored laboratorians to listen carefully to physicians when they have questions about test results. From her own experience, labs are variable in their interest in physician concerns.

Focusing on communication, Sluss takes a time-intensive, multilevel approach to ensure clinicians make the most of es-

tradiol testing. He meets with incoming medical house staff and discusses in detail the biochemistry of steroid hormones, re-views the different assay methods, their pros and cons, and presents case studies in which there are disparities between test results and clinical presentations. He also makes himself available to consult with cli-nicians as needed and spends considerable time reviewing test utilization. “I try to un-derstand ordering patterns and reach out to the people who have a high test volume to see if it’s meeting their clinical needs,” he explained.

Rosner extoled the power of these col-lective efforts. “If we insist as a commu-nity on accuracy-based standards, it can be done. It doesn’t happen overnight and it’s labor- and money-intensive but it’s what we should be doing.” CLN

ClinicalChemistryClinical Chemistry is pleased to announce a special upcoming theme issue on Women’s Health edited by Drs. Ann M. Gronowski, JoAnn E. Manson, Elaine R. Mardis, Samia Mora, and Catherine Y. Spong titled “Advancing Women’s Health: The Impact of Biomarkers and Genomics.” Clinical Chemistry, published by the American Association for Clinical Chemistry, is the most highly cited forum for peer-reviewed, original research in the fields of clinical chemistry and laboratory medicine.

The purpose of this issue is to highlight recent advances in biochemical and genetic markers used for the diagnosis, therapy, and preventive care of women during all stages of life. This issue will include diverse themes such as cancer, cardiovascular disease, osteoporosis, metabolic disease, normal and abnormal pregnancy, infertility, and infectious disease.

Clinical Chemistry invites authors to submit original articles related to women’s health to be considered for publication in this special issue. Potential topics of interest include:• Discovery and validation of novel biomarkers for the diagnosis, prognosis, and monitoring therapy of diseases that affect women including: cancer, cardiometabolic and/or cerebrovascular disease, bone disease, and autoimmune disorders• The genomics of ovarian, uterine, cervical, and breast cancer• The effect of gender on the risk factors and outcomes related to diabetes, obesity, and cardiovascular disease• Changes in the microbiome and biomarkers related to pregnancy­• Novel molecular diagnostic tools for pre-implantation genetic analysis• Non-invasive screening for fetal aneuploidies and other pregnancy outcomes• Novel biomarkers for the diagnosis of pregnancy-related disorders such as pre-eclampsia, ectopic pregnancy, preterm delivery, and gestational diabetes• Biomarkers for the diagnosis of infertility, menopause, and premature ovarian failure• The impact of infectious disease on the global health of women

Be a part of this exciting issue! Submissions must be received through our online submission system at http://submit.clinchem.org no later than July 1, 2013. Your cover letter should express your interest in submitting your paper for consideration for the Women’s Health theme issue. Journal guidelines for submission apply as described in the Information for Authors on the submission website.

call for papersAdvancing Women’s Health: The

Impact of Biomarkers and Genomics

2014 Special Issue

8 CliniCal laboratory news May 2013

Not only does the pituitary secrete TSH in a diurnal pattern, but many substances produced in the central nervous system, even in healthy euthyroid individuals, may enhance or suppress TSH production in addition to the feedback effect of thyroid hormone. Furthermore, although TSH levels rise and fall in response to changes in the concentration of free thyroxin (T4), individuals appear to have their own set-points, and factors such as race and age also contribute to variability in TSH levels. Al-terations of the normal pituitary response are also common in patients with a variety of illnesses.

Although laboratory measurement of serum TSH is an essential tool for diagnos-

ing and managing various thyroid disor-ders, the laboratory medicine community has long recognized that immunoassays used to measure the hormone are yet an-other source of variability in patients’ re-sults. These inter-assay discrepancies are now the focus of an international harmo-nization effort to improve the reliability of TSH results and their application in clini-cal guidelines (1). This article presents an overview of the issues and a discussion of

how laboratories can assist clinicians in us-ing TSH results to diagnose and manage thyroid disease.

Evolution of TSH Assay SensitivityThe sensitivity of TSH assays has increased substantially over the last several decades. The first TSH tests were competitive im-munoassays that used polyclonal antibodies; however, the analytical sensitivity of these as-says was not sufficient to differentiate hyper-thyroid patients with suppressed TSH from euthyroid individuals. Non-competitive im- munoassays introduced in the 1980s achieved this level of sensitivity, and in the 1990s, manufacturers further improved the assays by using monoclonal antibodies.

Today, we refer to the performance of these so-called second- and third-generation TSH assays in terms of functional rather than analytical sensitivity, meaning that imprecision is assessed over a concentra-tion range during an extended period of time. Functional sensitivity for TSH is de-fined as the level of TSH below which the imprecision of the assay exceeds a coeffi-cient of variation (CV) of 20%. By conven-tion, second- and third-generation assays

detect TSH with this degree of reproduc-ibility down to a level of approximately 0.1 and 0.01 mIU/L, respectively.

The majority of currently available TSH immunoassays are capable of third-generation performance. However, daily precision in this low range may not be as robust as it appears when functional sensi-tivity is initially evaluated (2). Furthermore, most laboratories probably do not regularly monitor performance in this low range, because it would require the use of in-house pooled sera. In addition, few, if any, proficiency programs regularly challenge laboratories at the limit of third-generation performance.

The major advantage of using a third-generation TSH assay is that precision at a higher level, 0.1 mIU/L, is markedly improved compared to earlier generation assays. In fact, most clinical guidelines rec-ommend 0.1 mIU/L as the cut-off for con-sideration of hyperthyroidism, followed by measuring patients’ total T4 and/or free T4. Third-generation assays also perform well in the range relevant for monitoring thy-roid hormone replacement therapy after thyroid ablation.

Measuring TSH is also important when hypothyroidism is suspected, because the inverse logarithmic relationship between TSH and T4 means that TSH levels will rise long before either T4 or free T4 concentra-tions fall. The upper limit of euthyroidism with first-generation TSH assays was ap-proximately 10 mIU/L, but with the intro-duction of second- and third-generation assays it fell to approximately 5 mIU/L. The most likely reason for this change was the reduced cross-reactivity afforded by the monoclonal antibodies used in the newer assays.

TSH Reference IntervalFor many years, most physicians have con-sidered TSH levels >10 mIU/L evidence of thyroid failure, and levels of 5–10 mIU/L evidence of mild or subclinical hypothy-roidism. During the past decade, however, there has been considerable debate about the correct upper limit of the reference in-terval for TSH.

Although there is a consensus that the lower limit of the euthyroid reference in-terval for TSH should be 0.2–0.4 mIU/L, experts disagree about the appropriate up-per limit. In 2002, researchers published

Thyroid-Stimulating Hormone Why Efforts to Harmonize Testing Are Critical to Patient CareBy JaMes D. Faix, MD, anD LinDa M. ThienponT, phD

Thyroid disease is common in the general population, and its prevalence increases with age. Because

the signs and symptoms of the disease often resemble other disorders, before initiating treatment

physicians need to determine whether the patient actually has thyroid disease or something else.

The test most frequently ordered to test thyroid function is thyrotropin, commonly referred to as

thyroid-stimulating hormone (TSH). Based on the functional interrelationship of the hypothalamus,

pituitary gland, and thyroid, TSH should be elevated if the thyroid gland is not producing adequate thyroid hor-

mone, and suppressed if it is producing too much (Figure 1). Today, however, we are beginning to realize that this

well-established paradigm for TSH synthesis and release is an oversimplification.

CLN’s

improviNgheaLthCare through LaboratorymediCiNe

series

CliniCal laboratory news May 2013 9

an analysis of thyroid function test results from a large survey of individuals represen-tative of the U.S. population (3). The study revealed that within a small standard error the mean TSH level in the general popula-tion is approximately 1.5 mIU/L. This find-ing prompted organizations to call for low-ering the upper limit of the normal TSH reference range. The National Academy of Clinical Biochemistry recommended 4 mIU/L, while the American Association of Clinical Endocrinologists set the upper limit at 3 mIU/L, and other groups went as low as 2.5 mIU/L. Many clinicians resisted these new limits, because they worried that a significant number of patients would be unnecessarily labeled as having thyroid dys-function, especially given the fact that there was no evidence that treatment of these in-dividuals would provide any benefit.

In 2007, researchers analyzed the data from the survey a second time to clarify the relationship of TSH and antibodies to thyroid peroxidase (TPO), a recognized marker of autoimmune thyroid disease (4). TSH levels correlated with anti-TPO posi-tivity, and the investigators asserted that reference interval studies would support the lower upper limit if such individuals, who probably have occult autoimmune hy-pothyroidism, were excluded. While some groups also have challenged this position, there is growing consensus that one TSH reference interval does not fit all.

The Special Cases: Pregnant Women, NewbornsDiagnosing thyroid dysfunction in preg-nant women has long been problematic. Pregnancy has a significant effect on thy-roid function, which changes over the course of gestation and makes assessment more difficult. The recent guidelines for

diagnosing and managing thyroid disease during pregnancy issued by the American Thyroid Association recommend trimester-specific reference intervals for TSH, as well as TSH targets for diagnosing and treating hypothyroidism during pregnancy.

Neonatal screening for congenital hy-pothyroidism is another special problem. Many programs have switched from an initial T4 screen with TSH confirmation of low T4 results to an initial TSH screen. Each screening program sets its own cut-offs, but standardization of TSH immunoassays would benefit from this approach.

TSH GlycoformsIn addition to inter-assay differences, evi-dence is now accumulating that there may be significant variability in the structure of the patients’ TSH molecules. TSH is a gly-coprotein hormone with a structure simi-lar to other anterior pituitary glycoprotein hormones. This group of hormones con-sists of non-covalently linked heterodimers: an α-chain common to all and a unique β-chain that specifies the hormone’s bio-logical activity. TSH has three sites where oligosaccharides may be attached, which can contribute up to 25% of the molecule’s mass. The pituitary actually releases a het-erogeneous mixture of TSH glycoforms that consists of molecules with various side chains (Figure 2).

TSH isolated from normal pituitaries contains primarily branched glycans with sulfated acetylgalactosamine (GalNAc) res-idues; however, the sera of patients with hy-pothyroidism contain TSH with branched glycans composed of sialic acid attached to galactose residues. The liver recognizes glycoproteins with the GalNAc sulfate sig-nal and removes them; therefore, sialylated TSH has a longer half-life. As a result, pa-

tients with hypothyroidism not only pro-duce more TSH, but also TSH glycoforms that circulate longer in blood (5). Since laboratories use the same assay to measure TSH in all patients, it is important to know how various manufacturers’ immunoas-says are affected by the presence of these different TSH glycoforms.

TSH Assays – The Status QuoIn recent years, laboratory professionals have increasingly recognized that TSH im-munoassays do not perform consistently. Data from the U.K. National External Quality Assessment Service proficiency testing program revealed poor correlation between observed method bias and the reference intervals recommended in the package inserts from various manufactur-ers, which are essentially equivalent. These observations suggest that standardization of TSH assays would be helpful for improv-ing patient care, particularly given the cur-rent discussions about lowering the upper decision limit for TSH in clinical practice guidelines.

All of these considerations contributed to the International Federation of Clini-cal Chemistry and Laboratory Medicine (IFCC) forming a Working Group for Standardization of Thyroid Function Tests (WG-STFT) in 2005. Now a full-fledged committee, the goal of this body—of which both authors are members—is to investi-gate practical options for standardization of thyroid hormones such as TSH, total and free T4, and T3. We first screened 200 apparently healthy individuals and selected a panel of 40 individuals to use as single-serum donors. For TSH, nine manufactur-ers representing a total of 16 immunoassays agreed to participate in the project.

In this initial investigation of TSH assay variability, we found that currently available TSH assays are precise. Within-run and total imprecision were 1.5–5.5% and 2.5–7.7%, respectively. Also, with regard to compara-bility, the mean results of most of the assays agreed within 10% of the target value. Be-cause no reference measurement procedure exists for TSH, we used the all-procedure trimmed mean as the target (6). Only three deviated by more than 10%, with two dif-fering by almost 40% in opposite direc-tions. Mathematical recalibration using the regression of each assay to the target value satisfactorily removed the individual assay biases, and the recalibrated results complied with the biological total error goal of 22.8%, except for one result in each of four assays (Figure 3). This outcome indicated that our approach, harmonization rather than stan-dardization (see below), would be possible. In fact, a subsequent study in which the manufacturers included their master cali-brators and were asked to recalibrate their assays was similarly successful (7).

Although different TSH glycoforms did not appear to affect assay performance in these initial studies, all of the samples were from apparently healthy individuals. Only two had suppressed TSH and only six had elevated TSH. In contrast, in a more recent study conducted in 2012, we evalu-ated TSH assays using more than 90 sam-ples from patients with thyroid disease, in whom TSH concentrations varied widely from 0.04–80 mIU/L. While the results of this study have not yet been reported, we can reveal that this method comparison confirms that current TSH immunoassays are “glycosylation-blind” and that recali-bration using the assay means significantly reduces individual assay bias.

Figure 1

Thyroid Function

The hypothalamus releases thyrotropin releasing hormone (TrH), which travels via a venous plexus to the anterior lobe of the pituitary gland and stimulates release of thyroid stimulating hormone (TSH). TSH then induces production of thyroxine (T4) by the thyroid. In response to the concentration of free T4, which influences the amount of triiodo-thyronine (T3) produced in each site, both the hypothalamus and the pituitary alter production of TrH and TSH, respectively. Elevated free T4 inhibits production, while low free T4 stimulates production.

Figure 2

Glycoforms of TSH

In euthyroid individuals, the pituitary gland produces a wide variety of TSH glycoforms. The majority have carbohydrate side chains terminat-ing with sulfated N-acetylgalactosamine or galactose, and some have internal fucosylation as well (arrows). The liver recognizes all of these moieties and removes TSH bearing them from circulation. By contrast, TSH glycoforms produced by hypothyroid patients have side chains lacking fucose and terminating with sialic acid residues. These are not removed by the liver and they have a longer half-life.

Adapted from reference 5; used with permission.

10 CliniCal laboratory news May 2013

TSH Assays – The Harmonized FutureAccording to the measurement paradigm, clinical laboratory results produced by assays claiming to measure the same measurand should be equivalent, regardless of where or how they are produced. This is especially important for patients who may visit dif-ferent healthcare systems during the course of their treatment. Ideally, this would be ac-complished by using calibrators traceable to a reference measurement procedure (RMP), which is a requirement of International Standards Organization (ISO) Directive 17511 (8). However, the ISO standard rec-ognizes that many measurands lack either a RMP or a commutable primary reference material for calibration, or both. When this is the case, the assay can not be standardized, but it might be possible to harmonize it.

Although investigators have been trying to develop one, no primary RMP for TSH is available. Current TSH methods all claim “traceability” to the World Health Organi-zation (WHO) TSH International Refer-ence Preparation 80/558, but all of them do not produce comparable results. This is probably because the WHO standard is not commutable, meaning that it does not produce the same numeric relationship within clinically meaningful limits for dif-ferent measurement procedures. This char-acteristic can be attributed to many factors. For example, laboratorians are familiar with the term matrix effect, which refers to alteration of either the sample matrix or the measurand during production of a reference material that makes one or both of them react differently in different mea-surement procedures. Therefore, it makes sense that a non-commutable measure-ment standard cannot be used for calibra-tion traceability (9).

The IFCC’s Committee for Standardiza-tion of Thyroid Function Tests (C-STFT) proposes harmonization of TSH using a new measurement standard: a panel of na-tive materials with values assigned by the all-procedure trimmed mean as the sur-rogate RMP (Figure 4). Full traceability to the existing WHO standard would be pre-served by transferring the unit of the WHO international standard to the first panel

of native sera by way of the all-procedure trimmed mean. However, from then on, the unit for immunoassays would be se-quentially transferred to follow-up panels by measurement in overlap, rather than to the next WHO standard, which would be-come a secondary calibrator. We also would like to emphasize that this harmonization approach will only be successful if certain medical, physiological, and analytical cir-cumstances outlined elsewhere are fulfilled (10).

Clearly, this harmonization effort re-quires several important components: a stable infrastructure for sourcing of clini-cal samples; effective communication to all stakeholders; and approval by regulatory authorities such as the U.S. Food and Drug Administration and the European Com-mission. The role of the manufacturers in this process is also critically important, and the C-STFT gratefully acknowledges their commitment to this project, not only for doing the measurements, but also for fund-ing the samples.

ConclusionsThe following recommendations should be formulated with regard to TSH testing: 1) until assay harmonization is implement-ed, assay-specific decision limits should be used in clinical practice guidelines and clinical studies; 2) third-generation assays should be clearly identified; 3) journal edi-tors should only accept studies that identify manufacturers’ assays and interpret study results using assay-specific cut-offs; and 4) proficiency testing/external quality as-sessment programs should use native sam-ples from single donations collected ac-cording to the Clinical Laboratory and Standards Institute C37-A protocol, but without filtration. CLN

ReFeRenCeS1. Miller WG, Myers GL, Gantzer ML, et al. Roadmap for harmonization of clinical laboratory measurement procedures. Clin Chem 2011;57:1108–17.

2. Reix N, Massart C, Gasser F, et al. Should functional sensitivity of a new thy-roid stimulating hormone immunoassay

be monitored routinely? Clin Biochem 2012;45:1260–2.

3. Hollowell JG, Staehling NW, Flanders WD, et al. Serum TSH, T4, and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutri-tion Examination Survey (NHANES II). J Clin Endocrinol Metab 2002;87:489–99.

4. Spencer CA, Hollowell JG, Kazarosyan M, et al. National Health and Nutrition Ex-amination Survey III thyroid-stimulating hormone (TSH)-thyroperoxidase antibody relationships demonstrate that TSH upper limit reference limits may be skewed by oc-cult thyroid dysfunction. J Clin Endocrinol Metab 2007;92:4236–40.

5. Donadio S, Pascual A, Thijssen JHH, et al. Feasibility study of new calibrators for thyroid-stimulating hormone (TSH) im-munoprocedures based on remodeling of recombinant TSH to mimic glycoforms circulating in patients with thyroid disor-ders. Clin Chem 2006;52:286–97.

6. Thienpont LM, Van Uytfanghe K, Beastall G, et al. Report of the IFCC Work-ing Group for Standardization of Thyroid Function Tests, part 1: Thyroid-stimulating hormone. Clin Chem 2010;56:902–11.

7. Thienpont LM, Van Uytfanghe K, Van Houcke S. Standardization activities in the field of thyroid function tests: A status re-port. Clin Chem Lab Med 2010;48:1577–83.

8. International Organization for Stan-dardization (ISO). In vitro diagnostic med-ical devices—measurement of quantities in samples of biological origin—metrological traceability of values assigned to calibra-tors and control materials. ISO 17511:2003

2003 ISO Geneva, Switzerland.

9. Miller WG, Myers GL, Rej R. Why commutability matters. Clin Chem 2006;52:533–4.

10. Thienpont LM, Van Houcke SK. Trace-ability to a common standard for protein measurements by immunoassay for in-vitro diagnostic purposes. Clin Chim Acta 2010;411:2058–61.

James D. Faix, MD, is direc-tor of clinical chemistry and immunology and clinical professor of pathology at Stanford University Medical

Center. He is also a member of the IFCC C-STFT. Email: jim.faix@standford.edu

Linda M. Thienpont, PhD, is director of the Laboratory for Analytical Chemistry and a member of the faculty of Pharmaceutical Sciences,

Gent University, Gent, Belgium. She is chair of the IFCC C-STFT. Email: linda.thienpont@ugent.be

Disclosure: The authors have nothing to disclose.

Acknowledgements: The authors are indebted to these manufacturers: Abbott, Beckman Coulter, bioMérieux, DiaSorin, Ortho-Clinical Diagnostics, Roche Diagnos-tics GmbH, Siemens Healthcare Diagnos-tics, and TOSOH Corporation. We also acknowledge the other members of the IFCC C-STFT: Michael Rottmann, Frank Quinn, Barnali Das, and Finlay MacKenzie.

Figure 3

Percentage Difference Plot for 16 TSH Immunoassays

The y-axis represents the difference of the mean of singlicate results from the all-procedure trimmed mean (APTM) as target value, and the x-axis displays the APTM. Graph A is the initial evaluation of the assays, and Graph B is after recalibration by the reverse regression equation from the method comparison data. The effect of recalibration, which re-moves most of the individual assay bias, is emphasized by highlighting the most negatively (blue circles) and positively (red triangles) biased assays. All other assays are denoted by the symbol X. The dotted lines represent the biological total error goal for TSH (22.8%).

Adapted from reference 6; used with permission.

Figure 4

TSH Harmonization Steps

The C-STFT’s proposed scheme for TSH alters the steps usually followed when attempting to standardize a clinical laboratory assay. Because there is no reference measurement procedure (RMP) for TSH and be-cause the WHO reference material does not appear to be commutable, a panel of single-donor samples will be used to establish target values for calibration and the all-procedure trimmed mean derived from a method comparison study including manufacturers’ master calibra-tors. This alternative approach has been referred to as “harmonization,” rather than standardization.

CliniCal laboratory news May 2013 11

In the fast-moving field of molecular diagnostics, the American College of Medical Genetics and Genomics (ACMG) recently released guidance on two emerging issues of impor-

tance: non-invasive prenatal screening for fetal aneuploidy (NIPS) and reporting in-cidental findings from whole exome or ge-nome sequencing.

In a policy statement on NIPS, ACMG noted that analyzing cell-free fetal DNA from maternal blood for fetal aneuploidy is likely the first of major steps toward fe-tal genome or exome sequencing. Prom-ising studies of this method have shown high sensitivity and specificity with a low false-positive rate. However, ACMG cited 10 limitations of NIPS, including: iden-tifying only three aneuploidies—leaving undetected about half those picked up by amniocentesis; not detecting chromosomal abnormalities such as deletions and dupli-cations; and having longer turnaround times than tests of maternal serum analytes. For these reasons, ACMG recommends that women undergo invasive testing to confirm positive NIPS results.

Pre- and Post-Test Counseling CrucialThe statement called on laboratory reports to state clearly that NIPS is a screening, not diagnostic, test, and to clarify the need for post-test counseling for patients with screen-positive or screen-uninformative results.

ACMG also issued recommendations for reporting incidental findings from whole exome or genome sequencing. This docu-ment, which followed a policy statement on clinical sequencing and was a year in the making, was crafted by a special work-ing group. The panel noted that the rapid advance of exome and genome sequencing has made it possible to use these methods in a variety of clinical settings, all of which have the potential to expose incidental findings that were not the reason for the sequencing. Considerable literature has explored both the utility and ethics of incidental findings in research settings, but little exists in the clinical realm, and is particularly lacking to support evidence-based recommendations. Even so, the working group arrived at con-sensus based on available evidence.

The panel compiled a list of 24 condi-tions and associated genes and variants that should be reported for incidental findings in all patients regardless of age, ranging from hereditary breast and ovarian cancer

(BRCA1/2) and familial hypercholester-olemia, to Lynch syndrome and hyper-trophic or dilated cardiomyopathy. The working group prioritized this list for con-ditions in which treatments or preventive measures exist and for which confirmatory diagnostic approaches are available.

The panel recommended against pa-tient choice about receiving the findings, noting that “clinicians and laboratory personnel have a fiduciary duty to pre-

vent harm by warning patients and their families about certain incidental findings and that this principle supersedes concerns about autonomy.” However, the authors also emphasized that incidental findings should be reported only to the ordering cli-nician, who could put them in the context of the patient’s personal and family medi-cal history, physical examination, and other factors. Laboratory reports of incidental findings should specify with “distinct lan-

guage” how the quality of those findings differs from the quality of molecular test-ing for the primary investigation.

Finally, the working group cautioned that its recommendations reflect current technology, and that the list of recom-mended conditions should be updated annually. CLN

The documents are available online, at www.acmg.net.

New Guidance on Genetic and Genomic TestingACMG Issues Advice on Cell-Free Fetal DNA Screening, Incidental FindingsBy Genna Rollins

Next Month in CLN

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• • • • • • • • •

Sequester Moves Forward, Taking Billions Out of Healthcare

With no action from Congress, reim­bursement to hospitals and other

providers under Medicare fell 2% in April, a cut of nearly $10 billion to healthcare as a result of the Budget Control Act of 20 II. With no deal in sight in Washington, the Centers for Medicare and Medicaid Ser­vices (CMS) said providers should expect to see cuts to claims dated on or before April 1, 2013. CMS emphasized that the sequestration claims payment adjustment will be applied to all claims after determin­ing coinsurance, any applicable deductible, and any applicable Medicare Secondary Payment adjustments.

Although beneficiary payments for de­ductibles and coinsurance are not subject to the 2% reduction, Medicare's payment to beneficiaries for unassigned claims will be, CMS said. If providers have questions

about how the claims adjustments will work, CMS is asking them to contact their Medicare claims administration contractor.

More information is available from CMS, www.cms.gov/Regulations-and­Guidance/Regulations-and-Guidance. htrnl.

• • • • • • • • •

President, Congress Look at Lab Cuts in Budget Battle

Both President Obama and an indepen­dent congressional advisory panel are

suggesting cuts to laboratory reimburse­ment to help reduce the deficit. A proposal in Obama's 2014 budget would cut lab re­imbursement under Medicare by an addi­tional $9.46 billion over 10 years, a drop of at least 14% over that time period. Adding this reduction to those already scheduled under current law would bring total cuts to the clinical laboratory fee schedule to an estimated 35-37% over the next 10 years,

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according to the American Clinical Labora­tory Association (ACLA).

"Clinical laboratories were already fac­ing cuts of 23 percent over 10 years from reductions enacted in the Affordable Care Act, a two percent rebasing, and seques­

tration-many laboratories that provide critical laboratory services for Medicare beneficiaries were already hard hit by these cuts;' said Alan Mertz, president of ACLA in a letter to Obama. "While the president's budget proposes cutting an additional 14 percent under the pretext of 'modernizing payments for clinical laboratory services; in fact, these reductions are so severe when added to cuts in current law that the ability of many laboratories to continue serving Medicare beneficiaries would be in doubt:'

Mertz noted in the letter that Medicare spent $S.9 billion on clinical laboratory ser­vices in 20 II-just 1.6% of total Medicare spending.

Separately, the Medicare Payment Ad­visory Commission (MedPAC), an inde­pendent advisory panel to Congress, also reminded legislators in its March bi-annual report that cutting the lab fee schedule could save Medicare money.

Specifically, MedPAC urged Congress to cut lab reimbursement as a way to pay for fixing the sustainable growth rate (SGR) system, Medicare's formula for paying phy­sicians. Established in 1996, the SGR for­mula is widely recognized as deeply flawed, and Congress has had to act each year to intervene to prevent massive cuts to phy­sicians under Medicare. MedPAC wants Congress to find a permanent fix to the SGR problem. MedPAC's proposal would mean a 10% cut to labs over 10 years, total­ing $21 billion.

MedPAC also expressed support for re­cent cost-saving programs initiated under the Affordable Care Act by the Centers for Medicare and Medicaid Services (CMS). The commission recommended CMS broaden programs such as penalties for ex­cessive readmissions and linking payments to quality outcomes. MedPAC also praised accountable care organizations, but noted adoption needs to move faster if Medicare's finances are to remain stable.

The report is available from the Med­PAC website, www.medpac.gov.

• • • • • • • • •

Use of Free Preventive Screening Tests Grows

Data show that about 71 million Ameri­cans in private health insurance plans

in 2011 and 2012 received coverage for at least one free preventive healthcare ser­vice, such as a flu shot, cholesterol test, or cancer screening, because of the Afford­able Care Act. The new data was released in a report from the Department of Health and Human Services (HHS). Additionally, an estimated 34 million Medicare partici­pants have received at least one preventive service, such as an annual wellness visit, with no out-of-pocket cost because of the healthcare law.

"Preventing illnesses before they be­come serious and more costly to treat helps Americans of all ages stay healthier;' HHS Secretary Sebelius said in a statement. "No longer do Americans have to choose

between paying for preventive care and groceries:'

Services that do not require cost sharing under the law include pap smears, choles­terol screening, screening HN and other sexually-transmitted diseases, and diabetes screening, as well as preventive care such as flu shots and meningococcal and pneumo­coccal vaccinations for high-risk adults.

More information is available from the HHS healthcare website, www.healthcare. gov.

• • • • • • • • •

Government Pushes More Aggressive Health IT Agenda

The Centers for Medicare and Medicaid Services (CMS) announced a stepped

up plan to accelerate adoption of health in­formation exchanges and electronic health records (EHR).

In 2013, the government is setting the goal of 50% of physician offices using EHRs and SO% of eligible hospitals receiv­ing meaningful use incentive payments by the end of the year. CMS is also increasing the emphasis on interoperability, promot­ing electronic information exchange across providers. It began this effort by issuing a request for information seeking public input about a variety of policies that will strengthen the business case for electronic exchange across providers to ensure pa­tients' health information follows them wherever they access care.

Meaningful use rules will continue to be implemented this year, defining what data must be able to be exchanged between health IT systems, including how data will be structured and coded so that providers will have one uniform way to format and securely send data.

According to CMS, EHR adoption has tripled since 2010, increasing to 44% in 2012, and computerized physician order entry has more than doubled since 200S.

The document is available on the Fed­eral Register website, www.ofr.gov.

• • • • • • • • •

Brigham and Women's Hospital Joins NINDS Parkinson's Biomarkers Search

The National Institute of Neurological Disorders and Stroke (NINDS) has

formed an alliance with Brigham and Women's Hospital (BWH) to accelerate the search for Parkinson's disease bio­markers. As part of this agreement, Cle­mens Scherzer, MD, a BWH neurologist and researcher, was awarded $2.6 million over 5 years to develop biomarkers and facilitate NINDS access to BWH's premier data and biospecimens bank for Parkin­son's. Scherzer and colleagues believe that "dark matter" RNA transcribed from so-called "junk" DNA contributes to the complexity of normal dopamine neurons and, when corrupted, Parkinson's disease. Working off of this theory, his team's project will initially search for these RNAs in the brain tissue of individuals in the earliest stages of the disease. Then, the team will look for related biomarkers in the bloodstream and cerebrospinal fluid of both healthy subjects and those with Parkinson's. According to Scherzer, such biomarkers will make Parkinson's clini­cal trials more efficient and less expensive, while also improving physicians' abilities to monitor patient disease progression and treatment response.

• • • • • • • • •

Thermo Fisher Buys Life Technologies

Thermo Fisher Scientific has inked a deal to acquire Life Technologies for

approximately $13.6 billion, plus the as­sumption of net debt when the deal clos­es in early 2014. Life's portfolio includes an extensive range of products and ser­vices in genomics and molecular and cell biology, as well as next-generation se­quencing capabilities. By merging these strengths with Thermo Fisher's offerings in analytical technologies and specialty diagnostics, the companies aim to ac­celerate results for proteomics, genom­ics, and cell biology customers through the convergence of life sciences tools and diagnostics. "The acquisition of Life en­hances all three elements of our growth strategy: technological innovation, a unique customer value proposition, and expansion in emerging markets;' said Marc N. Casper, president and chief ex­ecutive officer of Thermo Fisher.

• • • • • • • • •

OGT Signs Licensing Agreement for ICR Prostate Cancer Biomarkers

The Institute of Cancer Research (ICR) has granted Oxford Gene Technology

(OGT) a license to further develop and commercialize a new panel of diagnostic and prognostic microRNA biomarkers for prostate cancer. OGT and the ICR dis­covered these markers during a previous

3-year collaboration. Unlike present tech­niques for prostate cancer screening, which involve the biomarker prostate-specific antigen (PSA) and a digital rectal examina­tion, the biomarkers discovered by OGT and the ICR have a specificity of more than 90%. Additionally, they have the potential to not only identify prostate cancer but to also assess its aggressiveness. This will

allow physicians to tailor treatment based on prognosis. OGT plans to evaluate the biomarker panel in tissue, blood, and urine samples, with initial translation of the as­say to blood-based PCR testing showing promising results, according to OGT's press release.

tests in early-phase biomarker and drug development studies and clinical trials. PrimeraDx then has the rights to develop and market any resulting co-diagnostics, while the agreement also enables Quest to validate and offer clinical diagnostic infor­mation services based on these assays. The two companies initially plan to focus on cancer, although they may branch out into infectious diseases, genetics, and other con­ditions. The ICEPlex system picks up mul­tiple molecular changes due to its ability to simultaneously detect and quantify dispa­rate target types such as mRNA, miRNA, SNPs, DNA mutations, fusions, and DNA methylation.

• • • • • • • • •

Quest and PrimeraDx Ink Co-Diagnostics Deal

PrimeraDx and Quest Diagnostics have entered a non-exclusive diagnostics co­

development agreement that allows Quest to use the PrimeraDx ICEPlex platform to independently develop and use laboratory

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• • • • • • • • •

Rising Levels of Proteinuria Associated With Shorter Life Expectancy

Canadian researchers found a substan­tial and progressive decrease in life

expectancy associated with the presence and severity of proteinuria (Am J Kidney Dis 2013;61:644-8). The findings could increase recognition of the health burden associated with proteinuria and help pri­oritize programs or funding involving this prevalent condition.

The investigators analyzed medical re­cords for adults in Alberta, Canada with­out end-stage renal disease who had at least one outpatient measurement of pro­teinuria over a 4-year period. A cohort of 812,386 patients was then followed for at least 3 more years to identify all-cause mor­tality. The researchers estimated baseline proteinuria via urine albumin-creatinine ratio (ACR) or urine dipstick, with nor­mal by ACR being defined as <30 mg/g, mild as 30-300 mg/g, and heavy as >300 mg/g. Negative urine dipstick was consid­ered normal, trace or 1 + as mild, and 2+ as heavy.

Overall, 9.55% of men had mild and 2.03% had heavy proteinuria, while 7.48% and 1.22% of women had mild or heavy proteinuria, respectively. Life expectancy was substantially shorter for people with higher levels of proteinuria. Life expec­tancies for 40-year old men with normal, mild, or heavy proteinuria were 31.8, 23.2, and 16.6 years, respectively. Similarly, life expectancies for 40-year old women with normal, mild, or heavy proteinuria were 35.7, 25.2, and 18.2 years, respectively. The results mirror another recent study of the authors' in which they reported that lower levels of kidney function based on estimat­ed glomerular filtration rate were associat­ed with decreased life expectancy for both men and women.

• • • • • • • • •

Chimeric PCR Simplifies Testing for Huntington Disease

Researchers at ARUP Laboratories in Salt Lake City developed a chimeric,

or triple repeat, primed polymerase chain reaction (PCR) process for determining expression of the Huntington (HTT) gene that provides a simple strategy to differen­tiate normal from expanded CAG alleles, thereby reducing the number of samples

Next Month inCLN

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that still require expensive and labor­intensive Southern blot analysis 0 Mol Di­agn 2013;15:255-62).

Huntington disease is associated with abnormal expansions of the CAG repeat in the HTT gene. Samples that appear homo­zygous for a normal allele typically undergo additional testing to verify that PCR did not miss a CAG expansion. This approach might include testing the adjacent CCG

loci and amplifying over the CCG+CAG region. If the sample still appears homozy­gous, the next step is Southern blot analy­sis. In contrast, the chimeric method, which has been described previously in fragile X

screening, generates different sized ampli­cons due to multiple annealing sites on the template. In using the method in Hunting­ton disease, the authors found that it distin­guishes true homozygous alleles easily, and that only very large expanded alleles still require Southern blot analysis.

To test how well the chimeric assay per­formed, the authors analyzed 246 samples that previously had been analyzed by an­other method, had both homozygous and heterozygous samples, and a range of CAG repeats. The authors found that it correctly genotyped all samples, characterizing them all within ±1 CAG repeat. This gave the chi­meric method "perfect" concordance with the other method, with 100% sensitivity and specificity in detecting CAG repeats.

The investigators determined that all adult-onset cases of Huntington disease with <101 CAG repeats can be genotyped easily and correctly sized. In this way, ap­parent homozygous samples can be detect­ed reliably without additional CAG+CCG or Southern blot analysis.

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CliniCal laboratory news May 2013 15

Quidel Receives FDA Clearance for RSV + hMPV Test

FDA has cleared Quidel’s Molecular RSV + hMPV assay for detecting respiratory

syncytial virus (RSV) and human meta-pneumovirus (hMPV) and distinguish-ing between them. This duplex test shares a common extraction protocol with other Quidel molecular assays and can be run in batches simultaneously in the same multi-well PCR panel for customized multiplex-ing capability. The RSV + hMPV assay is Quidel’s third molecular PCR assay to receive 510(k) clearance. Previous Quidel tests cleared by FDA include the company’s molecular assays for Influenza A+B and hMPV, and its C. difficile assay in the Am-pliVue format.

FDA Approves Abbott Test for Birth Defects, Testicular Cancer

Abbott has received FDA approval for its Architect AFP test for detecting

serious birth defects and monitoring the progression of testicular cancer. Alpha-fetoprotein (AFP) is a biomarker that increases in mothers carrying a fetus af-fected by neural tube defects and in men with nonseminomatous testicular cancer. Abbott’s new test quantifies AFP levels in human serum, plasma, and amniotic fluid at 15 to 21 weeks gestation using chemi-luminescent microparticle immunoassay technology, and it runs on Abbott’s fully- automated Architect i2000, i2000SR, ci8200, and ci16200 analyzers.

Quidel and Life Tech Receive Simultaneous Clearances

FDA granted Quidel and Life Technolo-gies 510(k) clearances to market the

Quidel Molecular Direct C. difficile As-say with Life Technologies’ QuantStudio Dx and 7500 Fast Dx Applied Biosystems real-time PCR instruments. The Quidel Molecular Direct C. difficile Assay kit in-cludes an extraction-free, three-step sample

preparation process that bypasses the need for a heat step, timed step, and centrifuga-tion and generates results in less than 70 minutes.

The QuantStudio Dx real-time PCR in-strument includes a touch screen, reagent and sample tracking capabilities, and a lab-oratory information management systems interface. Its six-color feature allows users to multiplex six targets in one reaction. The instrument provides an optional test devel-opment mode that accommodates 96- or 384-well plates and can perform pathogen detection, gene expression analysis, SNP genotyping, copy number analysis, muta-tion detection, micro-RNA and other non-coding RNA analysis, and high-resolution melt analysis. It also accepts a proprietary qPCR microfluidics card that can perform 48 tests on eight samples simultaneously without liquid-handling robots and that can be used to design and implement cus-tom tests.

Beckman Coulter Gets FDA Nod for Hemoglobin A1c Assay

FDA cleared Beckman Coulter’s new hemoglobin A1c assay (HbA1c-) for

quantitative determination of HbA1c con-centration in whole blood. Designed for use with Beckman’s UniCel DxC System and Synchron/AU Hemolyzing reagent, the assay improves accuracy and preci-sion, according to the company’s press release. It also meets the latest accuracy grading from the College of American Pathologists and the recommendations of the National Glycohemoglobin Stan-dardization Program (NGSP) and Inter-national Federation of Clinical Chemistry and Laboratory Medicine. The assay is standardized and holds a certificate from the NGSP. Features of the new assay in-clude ready-to-use liquid reagents for A1c and total hemoglobin, a 5-level calibrator set, 30-day on-board reagent stability, and 7-day calibration stability. Additionally, it eliminates cross reactivity with HbA1a, HbA1b, acetylated hemoglobin, carba-

mylated hemoglobin, and glycated albu-min, as well as significant interference from hemoglobin variants HbS, HbC, HbD, HbE, and HbF.

Meridian’s Group A and B Streptococcus Tests Rated Moderate Complexity

FDA has re-categorized Meridian’s il-lumigene Group A Streptococcus and

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lar test for detecting Group A Strep phar-yngitis. According to the company’s press release, clinical studies show that the new test increased detection of positives by 53% over traditional culture in symptomatic patients. In addition, clinical studies also showed that broth enrichment followed by testing with illumigene Group B Strep-tococcus increases detection of Group B Streptococcus (GBS) positives by 29% over traditional culture. The latter test also fully complies with Centers for Disease Control and Prevention guidelines for the detection of Group B Strep.

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