Originally Published IVD Technology April 2005
Anniversary Essays
5. Molecular diagnostics
Genomic technologies have greatly expanded
the capabilities of clinical labs.
Gregory J. Tsongalis
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Gregory J. Tsongalis, PhD, is |
Since 1989, when the federal government announced the first efforts to sequence the entire human genome, research laboratories have been abuzz during an era of genomics that was unprecedented by any other in the history of biology. From the basic (and now more in-depth) understandings of biological mechanisms to the rapid advancements in technologies, the genomic revolution progressed at a pace that was difficult to keep up with. Clinically, the promises of an unveiled human genome included better diagnostic testing, more-accurate risk assessments, expanded preventive medicine practices, and better therapeutics.
Polymerase Chain Reaction (PCR)
While many laboratories used various blotting technologies as their initial foray into molecular diagnostics, the limitations of such technologies regarding sensitivity, turnaround time, and hands-on time became apparent. A major milestone in molecular diagnostics occurred in the early 1990s with the transition of polymerase chain reaction (PCR) technologies from the research laboratory to the clinical laboratory.
Many labs were validating or had already validated PCR for specific clinical diagnostic applications. PCR was first performed in multiple water baths and then in programmable heat blocks or thermal cyclers. It offered the increased performance that clinical laboratories desired. Through in vitro amplification of specific target sequences, labs could perform the qualitative detection of such targets within several days. Because of the nature of the PCR amplification process, the sensitivity and specificity of such reactions for genomic and microbial targets could not be equaled by any other technologies in clinical laboratories.
PCR rapidly became the method of choice for molecular diagnostic analysis. PCR was an open system that was easy to perform and allowed users to design assays easily and control reaction conditions. It also satisfied clinical turn-around times and was a universal technology that could be applied to multiple applications for genetic, infectious, and neoplastic disease testing.
However, while traditional PCR itself had gone through various modifications to improve performance and applicability, continual increases in test menus and volumes revealed PCR’s limitations. The most significant limitation was the need for both physical and chemical contamination controls. In addition, the post-PCR detection process often took longer than the amplification itself. Modifications to gel electrophoresis equipment, 96-well-plate liquid-detection systems, and blotting techniques were attempts to streamline the detection process.
Nonetheless, PCR remained a powerful tool that allowed for the amplification of specific yet small target sequences that could then be analyzed using a variety of methods. For example, as with Southern blot technologies, PCR products could be interrogated for mutations by using restriction endonucleases that would either cut or not cut a PCR product depending on whether the mutation created or destroyed an enzyme recognition site. This four-step analytical process consisted of the following: DNA extraction, PCR amplification, restriction endonuclease digestion, and gel electrophoresis.
Laboratories were also using a variety of other hybridization and blotting techniques. Other detection methods were on the horizon and promised to provide more-rapid detection combined with higher throughput.
Other Molecular Diagnostic Technologies
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Papanicolaou smear. |
Around the same time that PCR was becoming a widely accepted technology in research laboratories and making its way into clinical laboratories, the power of in vitro amplification was being recognized. Many IVD manufacturers began developing in vitro amplification technologies for high-volume clinical testing that would compete with PCR’s performance characteristics. In 1996, Abbott Laboratories (Abbott Park, IL) received the first FDA approval for an in vitro amplification technology. FDA approved Abbott’s ligase chain reaction (LCR) technology for detecting Chlamydia trachomatis and Neisseria gonorrhoeae.
Not only did Abbott introduce a new amplification chemistry, it also set the stage for the introduction of semi-automated platforms for nucleic acid detection with its LCx instrument.
Soon afterward, Roche Diagnostics (Indianapolis) launched the Cobas Amplicor for semiautomated PCR, followed several years later by the ProbeTec by Becton Dickinson (Sparks, MD) for strand displacement amplification analysis. Gen-Probe Inc. (San Diego) also developed an in vitro amplification technology, transcription mediated amplification, and launched the Tigris instrument that fully automates high-volume C. trachomatis and N. gonorrhoeae testing.
While the automation of such STD tests was at the forefront of the developing molecular diagnostics field, another wave of new advances in the diagnostic market was brewing. The HIV epidemic continued to spread worldwide and have a significant economic impact.
Despite the availability of different therapies with varying degrees of effectiveness, physicians were unable to monitor the success or failure of treatments in their patients. Chiron Corp. (Emeryville, CA) developed the first HIV-1 viral load assay, a new technology that was not PCR-based and could quantify the amount of virus in patient specimens. This branched-chain DNA (bDNA) signal amplification technology accurately measured levels of HIV-1 in patient plasma. Roche followed with a reverse transcriptase–PCR assay for quantifying viral load. In addition to the development of several generations of such assays, newer chemistries with automated instruments (e.g., the System 340 by Bayer Diagnostics [Tarrytown, NY] and the Roche Cobas Monitor) have resulted in viral-load assays that can detect as few as 50 copies of a virus per ml of patient plasma. Similar tests for hepatitis C, hepatitis B, cytomegalovirus, and others have been established.
Microarrays and gene chips were also being developed for molecular applications, from gene expression profiling to drug discovery. High-density arrays that exceeded 100,000 data points could evaluate complex biological processes known to be regulated by numerous gene and protein interactions. Such array technologies promised to unravel the underlying mechanisms of common yet genetically complex diseases such as cancer, cardiovascular disease, and diabetes. While Affymetrix (Santa Clara, CA) focused on the highest-density microchips for discovery research, Nanogen (San Diego) worked on technologies for clinical laboratories and developed an open-array system, allowing users to address electronically 100 positions on a cartridge that could be interrogated in just minutes.
Despite such efforts by IVD companies, many molecular technologies remained nothing more than glorified detection systems for PCR. In fact, most arrays offered too much information for the limited informatics systems of clinical laboratories. However, higher-throughput techniques that allowed for the genotyping of diseases causing genetic alterations needed to be streamlined into the daily workflow of clinical labs.
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The SmartCycler real-time thermal cycler system by Cepheid (Sunnyvale, CA). |
One such technology that addressed this need was the Invader assay by Third Wave Technologies (Madison, WI). This assay was an isothermal, linear amplification method that used 96-well plates and required minimal instrumentation. The Invader assays depended on cleavase enzyme activity and could distinguish between mutant and wild-type sequences. In addition to human genomic mutations, new treatments for HIV patients resulted in resistant viral strains. Automated DNA sequencing methods were used to detect such viral mutations associated with resistance to antiretroviral therapies. DNA sequencing, or the Invader assays, could also perform viral genotyping to determine subtypes.
Clinical Applications
Through the late 1990s, the molecular diagnostics field was aglow with new developments for qualitative and quantitative detection of infectious disease agents. With PCR still the technology of choice, IVD manufacturers developed devices to streamline the detection of PCR products. The introduction of semiautomated instruments for high-volume infectious disease testing was only a part of this effort. With the Human Genome Project approaching completion, mutation analysis was considered as important to genetic disease and cancer diagnostics as viral load was to infectious disease diagnostics. In addition to traditional restriction endonuclease digests and blotting, manufacturers developed PCR-mediated techniques such as the Readit assay by Promega (Madison, WI), which utilized DNA polymerase-mediated pyrophosphoro-lysis in an enzymatic detection reaction; automated DNA sequencing by Applied Biosystems (Foster City, CA), which used capillary gel electrophoresis systems; and the Nanochip by Nanogen, which represented the first array-based technology for clinical laboratories.
While the molecular field was initially focused on infectious diseases in early 2000, new insights into the mechanisms of human cancers emerged. Part of this was due to gene expression profiling. However, even before the production of such large quantities of data, the ability to look specifically at tumor cells in complex tissues via microdissection and fluorescence in situ hybridization (FISH) was a landmark in molecular oncology.
Similar to the HIV situation in which new therapeutics led to the need for viral-load assays, the development of the anticancer therapeutic Herceptin required a clinical laboratory test to determine a patient’s Her-2 receptor status. While many techniques in clinical labs could accomplish this task, FISH became the method of choice for evaluating Her-2 gene amplification and determining patient eligibility for Herceptin treatment. Even though FISH was previously performed in cytogenetics laboratories, developments of new chemistries for interphase FISH made it suitable for analyzing paraffin-embedded tissues and other traditional pathology specimens.
Other progressive developments in oncology and genetic testing have occurred during the past 10 years. Revealing the basic gene alterations associated with any given cancer required interrogating the DNA and RNA of malignant cells. Laser-capture microdissection proved to be a powerful tool in isolating pure populations of tumor cells for analysis. This technology eliminated the background signal produced in many assays by contamination from normal epithelial, inflammatory, and stromal cells.
In addition, the association of human pap-illomavirus (HPV) with cervical cancer was revisited. Widespread screening for high-risk HPV types from liquid cytology specimens became standard practice. Another signal amplification technology, hybrid capture by Digene Corp. (Gaithersburg, MD) became the only FDA-approved assay for HPV testing.
Molecular genetics also got involved in its first population-screening program for cystic fibrosis (CF). In 1997, the National Institutes of Health (NIH; Bethesda, MD) convened a consensus conference to address the need for a national CF screening program. The American College of Medical Genetics, in conjunction with the American College of Obstetricians and Gynecologists, recommended that all pregnant women should be offered screening with a minimum panel of 25 CF transmembrance conductance regulatory mutations. As a result of these newly developed guidelines, laboratories were obligated to develop both diagnostic and carrier screening mutation panels. Several assays, including the Roche CF Gold, the CFTR33 LiPA by Innogenetics (Gent, Belgium), and the CF v3 OLA assay by Abbott, were developed and offered as analyte specific reagents.
Improving Molecular Diagnostics
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The GeneChip Scanner 3000, with Hybridization Oven 640, GeneChip Fluidics Station 450, and computer workstation, by Affymetrix Inc. (Santa Clara, CA). |
With several higher-volume tests on menus and more lower-volume tests being required after the completion of the Human Genome Project, certain molecular diagnostic technologies still needed to be streamlined for clinical laboratory use.
Modifications to PCR such that amplification and detection occurred simultaneously in real time revolutionized the way clinical labs performed testing. This innovation led to more-rapid turnaround times for molecular testing by making analysis possible without the need to batch samples. Instead, laboratories processed and tested specimens as they arrived. Several versions of Applied Biosystems’ platforms, the Roche LightCycler, and the SmartCycler and GeneXpert by Cepheid (Sunnyvale, CA) offered real-time PCR. Labs could perform molecular testing immediately for infectious diseases, which would have a major effect on patient care. Laboratories could also perform smaller-volume tests for infectious diseases, oncology, and genetics on a random-access basis.
As many laboratories converted from traditional to real-time PCR platforms, another piece of the puzzle was still missing. While the majority of molecular testing no longer required extensive labor on the analytical end, processing rapidly became the bottleneck. IVD manufacturers responded with smaller nucleic acid–extraction instruments such as the KingFisher by Thermo Electron Corp. (Waltham, MA), and larger robotic instruments based on liquid-handling platforms by Tecan (Maennedorf, Switzerland). Qiagen Inc. (Hilden, Germany) followed with a series of automated extractors, as did Roche with the MagnaPure.
Conclusion
Many molecular diagnostic innovations have emerged during the past 10 years, with promises of continual upgrades, revisions, modifications, and new platforms. The molecular diagnostics field has already seen a shift from being very manual to the introduction of automation. As the number of molecular diagnostics applications continues to increase, the goal of providing clinically useful laboratory results will be a challenge with respect to technologies, financial responsibility, and education.
Copyright ©2005 IVD Technology
