Vol. 19 • Issue 4 • Page 38
Two positive trends are occurring in nucleic acid testing. One is to provide more information on each sample, including testing for multiple organisms in infectious disease. The other is to provide more timely results for personalized care.
Personalized medicine is greatly aided by automation that integrates sample preparation, amplification and detection, enabling clinical decisions in minimal time. Fully automated molecular diagnostic (MDx) platforms with rapid turnaround times have emerged; instead of testing for only one target (e.g., HIV), multiplex tests that analyze multiple targets that may explain patient symptoms are of greater clinical value.
Full Automation Defined
Fully automated MDx means that one instrument integrates sample preparation, amplification, detection and presentation of results. Sample preparation usually requires DNA and/or RNA extraction. Amplification requires target or signal amplification and may include reverse transcription if the starting target is RNA. Target ðamplification methods include the polymerase chain reaction (PCR), transcription-mediated amplification (TMA), loop-mediated amplification (LAMP) and/or strand displacement amplification (SDA). The most common signal amplification method is branched-chain DNA (bDNA). Detection is typically by fluorescence methods popularized by real-time PCR, including quantitative PCR (qPCR) and melting analysis.1In a fully automated MDx system, results can be electronic, printed or wireless, but must be produced without any user intervention after the initial introduction of the clinical sample to the system.
Advantages of Large Automated Systems
Reduced labor, ease of use and objective result presentation are primary advantages of MDx automation. While traditional MDx requires trained technologists for nucleic acid extraction, assay setup, troubleshooting and data analysis, automated platforms require little if any technical expertise. Minimal hands-on time reduces labor costs and on-board computer analysis eliminates subjective interpretation of results. Integration of the entire process in a closed system reduces contamination problems and consolidates diverse instruments into one device.
Several fully automated, larger footprint systems for nucleic acid testing have been marketed for years. Typically floor- standing instruments, these can run large batches of samples, focusing on throughput and cost efficiency. By way of example, such systems include:
• Gen-Probe Tigris®using TMA,
• Becton Dickenson Viper®using SDA,
• Siemens Versant®using bDNA and
• Roche COBAS®using PCR.
Automation is achieved by robotics that connect sample preparation, amplification and detection. Although the initial investment may be high, cost efficiency is achieved by the large numbers of samples typical of centralized laboratories.
Advantages of Smaller Systems
When the need for rapid results is critical, more distributed testing closer to the patient and faster turnaround times may be warranted. The potential for rapid nucleic acid diagnostics was first shown by real-time PCR on the carousel LightCycler® introduced by Idaho Technology, as an open platform in 1996 and subsequently developed by Roche.2
However, multiplexing was limited by the number of fluorescent channels. Higher level multiplexing and result reporting were developed by Autogenomics on the Infinity®platform using automated nucleotide extension and array hybridization. Another interesting development is the incorporation of microfluidics for amplification with detection by high-resolution thermal melting (Canon US Life Sciences). Amplification and detection are typically automated on these systems; sample preparation is not.
Completely automated nucleic acid testing that includes sample preparation, rapid turnaround times (30-90 minutes) and small instrument footprints are emerging (Table). Integrated instruments save space in increasingly crowded laboratories, reduce ðlaboratory errors in processing and can result in better patient care because of rapid turnaround for timely results. Those listed in the Table use disposable cartridges that supply reagents for sample preparation and contain processing in a closed system.
The Cepheid GeneXpert®was the first instrument in this space and some assays are FDA cleared as a CLIA moderate complexity platform. The system is based on real-time PCR, so multiplexing is limited by the number of fluorescent channels. A single sample is processed in each module, although multiple modules can be combined for greater throughput and the turnaround time is 45 to 60 minutes. Sonication is used for sample disruption.
The Becton Dickinson BD Max®implements HandyLab’s Jaguar platform, processing 24 samples in less than two hours. It is based on microfluidics and real-time PCR. Some FDA-approved BD GeneOhm assays will migrate to this platform.
The Iquum Liat™is a “lab-in-a-tube” platform that processes a single sample in 30 to 60 minutes. Also based on real-time PCR, it has the smallest footprint of the instruments detailed in Table 1. The Enigma ML or “minilab” is a modular real-time PCR system based out of the United Kingdom with results in <60 minutes.
The Idaho Technology FilmArray™is also a single-sample, fluorescence-based instrument. Detection is based on melting analysis. Clinical samples are disrupted by bead beating, followed by purification with magnetic beads and optional reverse transcription for RNA ðtargets. An initial multiplex PCR is then performed, followed by distribution of the sample over an array of 102 wells with nested PCR primers. After nested PCR, up to 34 targets in triplicate are detected by high-resolution melting analysis. Results are available in 60 minutes and the system is undergoing FDA trials.
Automated nucleic acid testing is nearing the point of care, although no system is CLIA waived and several are yet to be evaluated by the FDA. While the disposable cartridges used in rapid automated platforms are often complex and carry a higher cost than reference laboratory reagents, the overall cost of medical care is generally reduced by faster turnaround times, decreased labor and more informative results.
Dr. Wittwer is professor, Department of Pathology, University of Utah School of Medicine.
1. Wittwer CT, Kusukawa N. Nucleic Acid Techniques in: Burtis, C., Ashwood, E., Bruns, D. (Eds.), Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th edition, Elsevier, New York, 2005, pp. 1407-49.
2. Lyon E, Wittwer CT. LightCycler technology in molecular diagnostics. J Mol Diagn 2009;11:93-101.