BBI Contributing Editor

ORLANDO – The annual meeting and exhibition of the American Association for Clinical Chemistry (AACC, Washington), held at the Orange County Convention Center in late July in combination with the annual meeting of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), is the leading forum worldwide for introduction of new in vitro diagnostic products, as well as for discussion of new developments and trends in diagnostic testing. Major areas of emphasis at this year’s conference included new automated immunoassay systems, targeted at applications in mid- to large-size laboratories faced with increasing demand coupled with continued shortages of qualified labor; proteomics, which represents a major expansion in the range of protein analytes of interest in the laboratory compared to those measured today via immunoassay and special chemistry analyzers; and point-of-care testing, including new markers for stroke and expanded use of glucose monitoring in the hospital setting. New developments in cardiac markers also were described at the conference, including markers that promise to improve the early diagnosis of heart attacks to allow more rapid and efficient management of patients with chest pain.

As shown in Table 1, revenues in the global market for clinical diagnostic products are estimated at $27.2 billion in 2004, and are expected to grow about 7.6% in 2005 to $29.2 billion. High-growth segments of the market include molecular diagnostics and point-of-care testing, with the latter driven by continued double-digit expansion in the whole blood glucose testing segment. Growth in the molecular diagnostics market is the result of a number of factors, including continued expansion in infectious disease testing for patient diagnosis as well as disease screening, along with development of newer segments such as cancer diagnostics, pharmacogenetic testing, and prenatal testing.

New technologies such as microarrays are expected to play an increasingly important role in allowing large panels of markers to be used in diagnostics, which experts presenting at the conference believe will be important for analysis of complex diseases such as cardiovascular disorders, cancer and neurological disorders, as well as for the prediction of drug response. Nanotechnology also is being applied in the development of new diagnostic testing devices, both to increase the number of tests that can be performed on small blood samples as well as to improve detection sensitivity for immunoassays and molecular diagnostic tests. Suppliers in the clinical diagnostics market will need to continue investing in R&D to remain competitive, and also are likely to employ acquisitions as a strategy to enhance their technology portfolio.

Immunoassay automation developments

The market for immunodiagnostic products is one of the largest segments within clinical diagnostics, accounting for approximately one-third of the total market if immunoassay-based blood screening products are included. Nine of the top 10 clinical diagnostics companies participate in the segment, and worldwide more than 60 suppliers have a significant position in the market. Automated immunoassay systems account for most (80%) of the sales in the immunodiagnostics segment, although point-of-care testing products are exhibiting higher growth. As discussed by Kenneth Blick, PhD, of the University of Oklahoma Health Sciences Center (Oklahoma City) at AACC, the latest systems provide test results over a wide linear range, exhibit a high degree of stability, require sample volumes of much less than 100 uL, and provide analytical sensitivity allowing detection of less than 1,000 molecules of the target analyte.

The field has been revolutionized, from the standpoint of detection sensitivity, by the introduction of chemiluminescence. Adoption of automation in immunodiagnostics has been driven by the shortage of qualified laboratory technicians, by demands to reduce errors, and by requirements for reduced turnaround time in the central lab. For some applications, however, such as cardiac marker testing, labs have been unable to achieve the needed turnaround time using central lab testing, and have instead implemented point-of-care testing using devices such as the Triage Cardiac system from Biosite Diagnostics (San Diego) to further reduce turnaround time.

In many labs, improvements in workflow automation and turnaround time have allowed a movement away from stat testing, since all tests can be run as soon as the sample is available. Blick’s lab has achieved a 25% increase in immunoassay test volume while reducing the number of FTE’s in the lab, mainly through the implementation of automation.

Table 2 on below lists new automated immuno-assay systems that have recently been introduced or that are scheduled for launch within the next year. An important trend is the development of integrated chemistry/immunochemistry platforms. While integrated chemistry/immunochemistry systems have been available for a number of years, the most recent products offer capabilities in terms of sensitivity and throughput that make such systems truly practical for routine processing of test orders that include both standard chemistry analytes as well as analytes that are measured using high-sensitivity immunoassay techniques.

Examples of new high-performance integrated systems include the Abbott Laboratories (Abbott Park, Illinois) c16000, the Bayer (Tarrytown, New York) Advia 800 IMS, the Beckman Coulter (Fullerton, California) Access 2/UniCel DxC 600i/CTA platform, and the Dade Behring (Deerfield, Illinois) Vista. Suppliers also are addressing the smaller laboratory market with compact, dedicated immunoassay systems such as the Bayer Advia Centaur CP, the Roche (Indianapolis) Cobas e411, and the Abbott Architect i1000_SR.

Abbott’s new i1000_SR analyzer will eventually replace the AxSYM, which has more than 11,000 placements worldwide. While throughput for the i1000_SR will be similar to that for the AxSYM at 100 tests per hour based on a theoretical test mix, the new system will offer the advantages of continuous access and the ability to use the same reagents as the other Abbott platforms in the Architect immunoassay line.

Another trend is the ongoing conversion of the market to chemiluminescence labeling and detection technology, as exemplified, for example, by Abbott’s switch to chemiluminescence in its new ARCHITECT line; Dade Behring’s use of its LOCI technology in the Vista, and Roche’s decision to continue using electrochemiluminescence in its new analyzers now in development. As discussed by Blick, chemiluminescence now is the preferred technology for labeling and detection, due to its high sensitivity and reduced requirements for instrumentation as compared to fluorescence or enzyme-labeled chemistries.

Pace of proteomics discovery accelerates

An emerging area of potential applications for immunoassay systems is proteomics, which involves the analysis of human proteins for use as disease markers. Proteomics is not, strictly speaking, a new field, since many markers measured by immunoassay analyzers are proteins. The number of new protein assays introduced for clinical diagnostics has, in fact, been declining over the past few years, and the number of new assays introduced in the past two to three years is close to zero. Only 46 proteins are currently detected by the major analytical methods used in the clinical lab. However, an important limitation on the discovery of new protein markers until recently was the lack of discovery technologies that allow useful markers to be identified. Now, fueled by major advances in analytical technologies being employed in genomics and proteomics research, the pace of discovery of new protein markers has accelerated.

Experts estimate that there are in excess of 1 million proteins in plasma and cerebrospinal fluid, derived from only about 25,000 genes. Albumin comprises about 50% of protein present in blood by weight, and ten of the remaining proteins comprise 90%. Between 24 and 25 proteins account for 99% of all proteins by weight. The abundance of proteins varies by over 10 orders of magnitude, and, as discussed by Blick at the conference, there can be clinical significance at all levels. No diagnostic technique has yet been developed that can analyze proteins over such a wide dynamic range. Furthermore, there are about 90,000 different forms of secreted proteins, and approximately 500,000 forms of tissue proteins. About half of the variation in abundance of an individual protein is due to genetics, and half is due to disease processes. Typically, there is more variation in protein levels between individuals than within an individual over time, except in some disease states.

An important next step is to develop technologies that will allow protein markers discovered in research programs to be developed for use in clinical diagnostics, in order to tap into the large number of proteins that now are not used for disease diagnosis, but that may represent useful new clinical markers.

The presumed route to implementation of a new proteomic marker as a diagnostic test is to first identify the marker using a technique such as mass spectrometry, and to then develop an immunoassay that allows rapid, low-cost measurement of the marker in the clinical lab. One limitation, however, is that mass spectrometry typically has lower sensitivity than immunoassay, so many low-level markers with potential clinical utility are not identified during screening. In addition, some molecular mutations and glycosylations are not detected by immunoassay.

Some clinical labs have already begun using mass spectrometry for analyzing proteomic markers, but test volume is low, typically less than 10 tests per week. In addition, techniques such as SELDI-MS are highly susceptible to pre-analytical factors, making it challenging to use them for high-volume clinical testing. As a result, many proteomic tests are likely to use existing immunoassay technologies when commercialized for the clinical lab, since today’s immunoassay methods have more than adequate sensitivity. In some cases, the lower selectivity of immunoassays may present a problem, requiring developers to use mass spectrometry.

Mass spectrometry also is expected to play a role in clinical proteomic testing in combination with emerging microarray technologies, particularly in applications that involve measurement of large numbers of protein markers simultaneously. At least one company has announced a partnership agreement to commercialize protein microarray/mass spectrometry tests for clinical diagnostics. Ciphergen Biosystems (Fremont, California) reported an agreement with Quest Diagnostics (Lyndhurst, New Jersey) just prior to the AACC conference under which the two companies will form a strategic alliance to develop and commercialize proteomic diagnostic tests based on Ciphergen’s SELDI ProteinChip technology. The agreement also included an equity investment by Quest in Ciphergen, and funding of development activities by Quest.

Ciphergen had previously formed a diagnostics division that is engaged in discovery of new protein markers, focusing primarily on new cancer markers. Major efforts are also under way to discover new markers for neurological diseases (particularly Alzheimer’s disease), cardiovascular disease and infectious diseases, as well as markers for prediction of treatment response and adverse drug reactions, and markers for prognosis and risk stratification in various diseases. While the tests that will initially be commercialized through the Quest partnership will be reference laboratory assays that will not require 510(k) or PMA applications, Ciphergen expects to eventually develop in vitro diagnostic products that will be cleared via the 510(k) process and marketed to hospitals and other clinical labs.

Another unique new test that involves a combination of protein and DNA analysis was recently described by Cangen Biotechnologies (Bethesda, Maryland) and Olympus (Tokyo). The two companies are developing a high-throughput test for the early detection of lung cancer that will employ microarray technology from Olympus. Cangen is developing a new technology platform that combines mass spectrometry with micro-satellite DNA analysis. The goal of the partnership is to develop a cost-effective test to detect lung cancer at an early stage.

Rapid growth for cardiac markers

Cardiac markers are one of the fastest-growing segments of the immunoassay market, and were the focus of several sessions at the AACC conference. New markers are under development both for improved diagnosis of acute coronary syndromes, as well as for improved risk assessment in cardiovascular disease. The role of cardiac markers has evolved considerably over the past few years, as discussed by Elliott Antman, MD, of Harvard Medical School (Boston) at an AACC plenary session. He said that 35 years ago, the diagnosis and treatment of acute coronary syndromes was based on erroneous concepts of the cause of coronary heart disease.

Recent improvements in the understanding of coronary artery disease have increased the utility of cardiac markers by allowing physicians to accurately interpret the meaning of test results, and researchers have used new knowledge of the basis of coronary disease to guide them in the discovery of improved markers. For example, troponins have played a central role in the re-definition of myocardial infarction in new guidelines issued by the American College of Cardiology (Bethesda, Maryland) and the European Society of Cardiology (Sophia Antipolis, France).

Another new marker, high-sensitivity C-reactive protein (hs-CRP), is an indicator of vascular inflammation, now known to be an important precursor to an acute coronary event. The marker is proving valuable in assessment of risk for acute coronary events, particularly for prediction of the risk of a second event in patients who have had a recent episode. Researchers are now investigating the use of combined markers, such as Troponin I, hs-CRP, D-dimer and BNP (B-type natriuretic peptide), to improve prediction of outcome for patients who have had an acute coronary event. Such panels can provide information on the key factors involved in coronary events, including myocardial tissue damage, vascular inflammation, hemostasis, and cardiac stress.

According to Antman, future developments could include the use of marker levels to provide a more detailed indicator of risk, as well as increased use of marker panels to provide a more comprehensive assessment of the factors involved in acute coronary syndromes. He said he also believes that assays for troponin fragments could potentially prove valuable in improving coronary disease risk stratification.

Another new cardiovascular disease risk marker was described at the AACC conference by Quantimetrix (Redondo Beach, California). The company has just submitted a new HDL subfractionation method for FDA marketing clearance that will allow testing to be performed in any laboratory. At present, HDL subfraction measurements, which provide a more accurate indication of the risk of myocardial infarction than total HDL levels alone, are performed in specialized reference labs, such as Berkeley HeartLab (Berkeley, California). Quantimetrix already markets the Lipoprint assay, which provides LDL subfraction data. The Lipoprint analyzer is priced at $18,000, and consumables are priced at $1,500 for a 100-test kit. The new test will add HDL subfraction testing capability to the Lipoprint system.

While high HDL has typically been considered to be a marker of good prognosis, Quantimetrix has found that certain subfractions of HDL are correlated with poor outcome. Thus, two patients with the same total HDL level can have significantly different risk based on the distribution of HDL subfractions, which relate to differing size of the HDL particles that exist in blood. Research suggests that small HDL particles may actually behave like LDL with respect to their lipid transport capabilities. The analysis provides data on 10 different HDL subfractions in a color-coded format. The HDL subfraction data can be used by physicians to select therapy, since statin treatment alone does not necessarily alter the high-risk subfractions, and other treatments such as niacin combined with diet and exercise may be required.

A new cardiac marker attracting considerable attention within the diagnostics community is whole blood choline. As discussed by Oliver Danne, MD, of University Hospital Charite (Berlin, Germany), at a symposium on cardiac markers sponsored by Abbott Diagnostics at the AACC conference, free choline was originally found to be associated with unstable angina in nuclear magnetic resonance studies conducted on blood samples. Choline levels where found to be elevated by about two-fold in unstable angina patients vs. patients with stable angina. The studies indicated a link between the release of choline into the bloodstream and plaque instability, as well as platelet activation.

Subsequently, Danne found that choline is a marker of non-occluding white thrombus, but not of occluding red thrombus. The predictive value of choline for myocardial infarction was found to be superior to that of either CKMB or myoglobin, two widely used markers for early detection of an infarction. The release pattern of the marker varies considerably between patients, with some not exhibiting any elevation in an acute coronary event. However, there is a high correlation between the level of whole blood choline and the need for intervention, according to Danne.

In addition, there is a three- to five-fold increase in risk of subsequent events if both whole blood choline and Troponin I are elevated, and an increased risk if elevated choline is detected in the absence of Troponin I elevation. In a study of seven different risk markers that included whole blood choline, BNP and Troponin I, whole blood choline and BNP were found to be the best predictors. Danne said he believes that whole blood choline is a valuable new marker that should be added to the panel of tests used to routinely diagnose myocardial infarction and predict risk for subsequent events. Ideally, point-of-care choline tests should be developed, he said, to take maximum advantage of the ability of the marker to provide an early indication of an event.

Hospital-based POC testing expands

Point-of-care (POC) testing is, in fact, becoming an increasingly important segment of the clinical diagnostics market, not just for cardiac markers but also for other analytes such as glucose, infectious disease markers, sepsis markers and markers of stroke. One rapidly growing application in the hospital setting is tight glycemic control (TGC), which involves the use of insulin infusion and intensive glucose monitoring to maintain blood glucose levels within a narrow range centered around 90 to 100 mg/dL. As discussed by Anthony Furnary, MD, of Oregon Health and Science University (Portland), who developed the Portland Continuous Intravenous Insulin (CII) Protocol, TGC can result in a 50% drop in mortality for diabetic coronary artery bypass graft patients. There are 165,600 diabetic cardiac surgery patients in the U.S. alone.

The benefit is not limited to diabetic patients, since even non-diabetics achieve a significant drop in mortality. TGC also results in decreased incidence of other adverse outcomes such as polyneuropathy, renal insufficiency, atrial fibrillation and anemia requiring transfusion. Benefits also are observed in patients with traumatic brain injury, in stroke patients, and in patients with sepsis. Although there is some added cost associated with TGC due to the higher number of glucose tests that must be performed at the bedside (testing is performed at intervals as short as one-half hour), the incremental cost of $138, as determined in a study of more than 5,000 cardiac surgery patients conducted by Furnary, is insignificant when compared to the savings resulting from reduced length of ICU stay and a lower rate of adverse events.

In Furnary’s study, the net savings per patient was $4,556. That figure translates to a savings of $500 million in annual healthcare costs in the U.S. for cardiac surgery patients alone. Furthermore, there have been essentially no adverse events attributable to TGC. The rate of hypoglycemia in patients treated with TGC is about 5%, and those cases can easily be managed with a simple glucose bolus.

There are certain issues related to whole blood glucose monitoring technology and its adequacy for use in TGC protocols. Since patients in the ICU often have abnormal blood parameters, such as low hematocrit and low oxygen saturation, some glucose meters may not provide sufficiently accurate readings to allow TGC to be implemented properly. Consequently, Furnary recommends that clinicians select the glucose testing system carefully, with particular emphasis on accuracy within the target range. The Abbott Diabetes (Alameda, California) FreeStyle system has performed particularly well in accuracy studies, for example, because its use of coulometry rather than amperometric technology renders it less susceptible to changes in pO₂, and a hospital-grade version of the FreeStyle is now in development.

Another new system with applications in TGC is under development by Nova Biomedical (Waltham, Massachusetts). The Nova system incorporates a test strip that can simultaneously measure four analytes, including glucose, hematocrit and oxygen, as well as interferants such as acetaminophen. The strip requires a 1 uL blood sample, and produces a reading in four seconds. Due to its ability to reject many common interferences, the system promises to be useful for TGC in neonates, dialysis patients, and in patients undergoing oxygen therapy. Nova is submitting a 510(k) clearance application and expects to release the product in late fall of this year.

In the future, continuous glucose monitoring systems, such as the Guardian system from Medtronic Diabetes (Northridge, California) that recently received FDA clearance for consumer use, may play an important role in TGC in the hospital setting, particularly since continuous monitors could eliminate some of the workload issues that TGC creates for ICU nurses. At present, workload issues are one factor limiting the widespread adoption of TGC in the U.S., although adoption is nevertheless growing rapidly.

According to a survey conducted by LifeScan (Milpitas, California) and presented at the AACC meeting, the number of hospitals using TGC has more than tripled over the past year. In July 2004, at least 90 LifeScan customers had implemented TGC. By July 2005, 237 additional hospitals had adopted the protocol, for a total of nearly 330. At Duke University Medical Center (Durham, North Carolina), as related by John Toffaletti, PhD, at the AACC gathering, the volume of POC whole blood glucose testing has increased from 2,500 to 25,000 per year. Given the clinical and cost benefits of TGC, it is likely that all hospitals in the U.S. will implement TGC for at least some portion of their patients over the next few years.

Use of other types of POC tests also is expanding, although more slowly than expected in some cases. For example, POC cardiac marker tests are now available from a number of suppliers, including Biosite, Abbott Diagnostics (on its i-STAT POC analyzer), Response Biomedical (Burnaby, British Columbia), and Roche Diagnostics. However, according to Fred Apple, PhD, of Hennepin County Medical Center (Minneapolis), who discussed POC cardiac marker testing at a symposium on cardiac markers sponsored by Abbott Diagnostics, only 15% of hospitals are using POC cardiac markers based on the most recent report from the College of American Pathologists (Chicago).

Apple has found, however, that the use of POC cardiac marker testing in the emergency department in his institution has a major positive impact on patient triage. Many speakers at the AACC conference agreed that POC cardiac marker testing is of value, and in a number of cases it provides the only solution for meeting the turnaround time requirements of physicians who treat myocardial infarction patients.

Microalbuminuria screening development

Another important development in POC testing was reported by HemoCue (Lake Forest, California) at an AACC press conference. HemoCue has introduced a new POC urine albumin system for microalbuminuria screening. Microalbuminuria is the primary indicator of early renal failure in diabetic patients, and screening for microalbuminuria is now recommended by the American Diabetes Association (Alexandria, Virginia) for all Type 1 diabetics at five years following initial diagnosis, and upon initial diagnosis for Type 2 diabetics. The new Hemocue test requires only 15 uL of urine and a result is obtained in 90 seconds. The test provides a quantitative result over the range of 10-150 mg/L, and is CLIA-waived.

Early detection of renal disease can have important ramifications, since appropriate treatment at an early stage can effectively delay the onset of renal failure until the patient dies of another cause. Microalbuminuria is typically detected years before creatinine levels become elevated in diabetic patients, making it a highly sensitive early indicator. As shown in Table 3, microalbumin test volume in the U.S. has grown substantially over the past few years, and volume is expected to increase at over 22% per year through 2009.

The introduction of improved POC test methods for microalbuminuria is expected to result in an increase in screening due to the greater convenience of office and clinic-based testing. HemoCue is investigating partnerships with major suppliers of products for diabetes management, such as pharmaceutical companies, to expand its marketing presence.

A competing POC microalbumin test is marketed by Bayer Diagnostics, which has a Microalbumin/ Creatinine test cartridge available for its DCA 2000 POC testing system. The Bayer test offers a seven-minute turnaround time and requires a 40 uL urine sample.