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Published: August 31, 2011
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Tools for Molecular Diagnostics

From IVD Technology's 2011-2012 Buyers Guide

By: Frederick Eibl and Romain Prieur

The molecular diagnostics industry is growing rapidly, thanks to the inherent accuracy, sensitivity, specificity, and quick turnaround of molecular techniques.The molecular diagnostics industry is growing rapidly, thanks to the inherent accuracy, sensitivity, specificity, and quick turnaround of molecular techniques. In addition, because of the accuracy, sensitivity, and specificity, laboratories can obtain valid results from very small amounts of specimen. This is useful for the forensic area, but also for detecting very low concentrations of target material, enabling clinicians to detect disease at a very early stage.
As a result, a growing number of companies are developing molecular tests for specific gene sequences and hoping to sell them to diagnostic laboratories, but those developers may not all understand the need for additional tools that support the entire workflow required to go from sample to result.
This article presents the types of technologies that are necessary to implement molecular diagnostic testing as well as some of the tools on the horizon that have the potential to revolutionize molecular diagnostics in the same way that PCR has.

For years, the core technologies in most molecular diagnostic laboratories have been focusing on methods that detect a specific, relatively short section of DNA or RNA that can diagnose an infectious disease, identify specific gene variants that affect drug metabolism, or test for genes associated with diseases such as cancer. At the heart of such tests lie amplification technologies such as real-time quantitative polymerase chain reaction (PCR), transcription-mediated amplification (TMA), target amplification, and signal amplification. Sanger sequencing and DNA fragment analysis or sizing applications using capillary electrophoresis are also key technologies for the molecular diagnostics laboratory, and they often also include an amplification step in the procedure. To successfully implement tests that can use DNA and RNA to diagnose disease, molecular diagnostics laboratories must implement well-defined work flows that allow the generation of robust, validated results, and there are specific tools that can help streamline each step of the work flow. The basic workflow is as follows:

1. Sample collection and preparation: extraction of the genetic material to be tested from the specimen.
2. Amplification: once the genetic material has been isolated, it must be amplified to a point where it can be detected in order to make diagnostic calls.
3. Detection: once enough target material has been generated, light-dependant sensors will be able to read signals that correspond to the target materials to be detected. The signals might be single or multiple to allow detection of multiple targets in one reaction (i.e., a multiplex assay).
4. Data analysis: analysis of the readout of the signals generated during the detection phase. Transforms those into ready-to-interpret information for the laboratory personnel to finally provide a diagnostic answer to the clinician.

Sample Preparation. Sample preparation, which includes sample extraction, is the first step in any molecular test. Sample preparation isolates nucleic acids (DNA or RNA) of interest and removes any potential inhibitors in the patient sample that could interfere with the downstream processes. It also concentrates the nucleic acids and increases the ability to detect very low concentrations of a target.
The type of patient sample heavily influences sample preparation methods. Sample types commonly tested in molecular laboratories include whole blood, serum, plasma, urine, stool, sputum, swabs, fresh frozen tissue, and formalin-fixed paraffin-embedded (FFPE) tissue. Depending on the type of patient sample tested, different sample preparations are required to successfully extract the nucleic acids without damaging the genetic material.
Different extraction kits can be tailored to patient sample type and to the nucleic acid source. For instance, if the nucleic acid comes from bacteria as opposed to viruses, a different extraction method should be used because the chemistry for extracting bacterial versus viral DNA could be different and there may be different steps involved to obtain the optimal sample. Sample extraction kits can even be specific for DNA versus RNA, thereby increasing efficiency for a particular type of nucleic acid.
Although some molecular diagnostic laboratories are still performing manual sample preparation methods, many laboratories are using semiautomated or fully automated sample-preparation instruments. Automation helps achieve more-consistent results in laboratories by eliminating the operator-specific variability associated with manual methods and reducing hands-on time. This allows for more productivity and can increase laboratory throughput.
An important area of focus for molecular diagnostic laboratories is streamlining of procedures and  ensuring superior quality control (QC). Proper QC practices are vital in ensuring accurate test results and delivering quality patient care. Quality control on the instrument begins at the sample-extraction step with technologies that verify that extraction is working properly. Commercially available products exist to aid the laboratory in the assurance of reliable results. An example of this is an extraction control that mimics the required steps necessary for patient samples and follows the same work flow to achieve the most accurate results.
In the case of quantitative assays, where an important result is a calculation of the number of copies of a genetic sequence, it is critical that the standards that are used to calibrate the assay also go through extraction for the following reason: extraction always results in some DNA or RNA loss. While it may be minimal, PCR’s exponential amplification can make small losses from extraction result in significant shifts in copy-number results. Running standards through the extraction step will ensure that sample losses due to extraction will be accounted for, and the patient sample results will be more accurate.
Amplification and Detection Platforms and Assays. Following sample extraction, target amplification followed by detection are the next steps in the molecular diagnostics  work flow. Amplification methods such as TMA are isothermal and do not require temperature cycling. PCR requires multiple cycles of heating and cooling to provide the nucleic acid template for replication. Tools that are required for this step include assay reagents and an instrument that enables the enzyme-based amplification of genetic material.
Assay reagents may consist of a number of materials including nucleotides, primers that are necessary for enzymes to amplify the DNA, probes that detect specific nucleic acid sequences, enzymes that power the reaction, buffers, and more.
Thermal cycling amplification occurs in a thermal cycler, and, depending on the technology, detection may occur after amplification (such as with single-nucleotide extension assays). In the case of real-time PCR, short tandem repeat (STR), or microsatellite assays, detection can occur simultaneously with amplification. For example, on the Applied Biosystems 7500 Fast Dx Real-Time PCR instrument for use with Center for Disease Control’s rRT-PCR flu panel, during the amplification step, a detector monitors for the signals that target-specific probes produce as the reaction progresses. With fragment-sizing assays, such as STR analysis, fluorescent dye-labeled primers are used during the amplification step, enabling easy detection and fragment sizing by capillary electrophoresis after amplification.
Most amplification and detection instruments can detect multiple signals at different wavelengths. For example, the aforementioned real-time PCR instrument can detect up to five color excitations at one time. With these capabilities, many molecular diagnostic laboratories are implementing multiplex assays. These multiplex tools can amplify and detect multiple targets for the same sample in a single tube. They can also detect several different targets in different tubes during the same run. The costs per reaction in multiplex assays can be significantly lower than for multiple assays that only detect a single target and are therefore attractive to a molecular diagnostic lab.  
For experienced molecular diagnostic laboratories, homebrew (laboratory-developed) tests may seem like a less expensive option to developing diagnostic assays. However, it requires additional time, money, and resources to establish the assay parameters necessary for validation, such as sensitivity, specificity, linearity, precision, and accuracy. This work can take several months, and laboratories often can save money and time by purchasing an FDA-cleared or -approved diagnostic assay from a manufacturer and getting that test online sooner.
Whether a lab chooses a homebrew or an FDA-cleared or approved test, independent quality control materials are extremely important in that process. Previously tested patient samples can be used to perform some of the characterization experiments, but in general, the availability of patient samples is very limited. By utilizing standards and controls from a provider that manufactures consistent and traceable quality materials compliant to ISO 17511, laboratories are not only assisted in setting up their assays but also in ensuring that assay is performing as expected over time and reported results are standardized across different molecular diagnostic laboratories. Some of these standards and controls are 510(k) cleared and provide an extra level of confidence and quality to the laboratory director.
Software Tools. Following amplification and detection and after discarding the amplified products, technicians are left with data only. Amplification and detection instruments have specific software programs that interface with the detectors in the instrument and collect data either during the thermal cycling process in the case of real-time instruments, or following a run in the case of end-point PCR and sequencing or DNA fragment analysis applications using capillary electrophoresis. A technician must review the analyzed data before results can be reported, and the amount of data manipulation that can be done depends on the instrument, analysis software, and application. Some instruments simply report values while others allow adjustments such as base-lining the amplification curves. For laboratory-developed tests, most data can be exported into spreadsheet programs for analysis. The analysis may include checking that the run meets validity criteria, eliminating outliers, and determining statistical values such as means and log values to interpret results.
In the end, the molecular diagnostic lab will have a report with either qualitative or quantitative results for the patient, depending on the assay.
Other software tools, such as EDCnet, allow laboratories to upload data and share molecular results of a known reference material for the same tests, and then compare their results to what other laboratories are observing for the same material (known as peer group comparison). This feature adds another layer of quality control to the laboratory in delivering quality test results to the clinician.
Total Solutions. Amplification-based diagnostics in molecular laboratories will remain a mainstay for disease diagnosis and pharmacogenetic studies, but more laboratories are investing in total solutions that go from sample-in to result-out with a single instrument. Combining sample preparation, assay set-up, amplification and detection, data analysis, and results reporting into fully automated instruments is the constant challenge for diagnostic suppliers. Some manufacturers have successfully accomplished this. For example, Cepheid’s GeneXpert and Gen-Probe’s Tigris system have integrated extraction, amplification, and detection on a single platform. Others have combined some steps such as sample preparation and assay set-up; however, the amplification and detection steps require separate instruments. The closer manufacturers get to total solutions, the higher the value proposition the system offers to the molecular diagnostics laboratories.

The Future of Molecular Tools
Molecular diagnostics is widely used in blood screening and infectious diseases, and to a lesser extent, genetic screening. Current molecular applications include viral-load monitoring to assess efficacy of anti-viral drug therapies, diagnosis of sexually transmitted diseases, and genotyping.
While genetic screening is a smaller area for molecular diagnostics, it is rapidly growing in the areas of oncology, gene expression analysis, companion diagnostics, and pharmacogenomics. These trends are in part of a personalized medicine approach to patient care, where the tools of technology are converging into the realm of diagnostics.
Prognostic evaluations of genetic mutations to determine susceptibility to disease conditions continue to generate interest from diagnosticians. Non-symptomatic patients at risk for certain genetic diseases or recovering from cancer therapy may undergo genetic screening to proactively manage their condition.
With the proof of the clinical utility of genetic biomarkers like human epidermal growth factor receptor 2 (Her2), epidermal growth factor receptor (EGFR), and V-Ki-ras2 Kirsten ras sarcoma viral oncogene homolog (KRAS) to predict which patients are likely to respond to drug therapies (Herceptin, Erbitux, and Vectibix, respectively), FDA has required changes in drug labeling, directing clinicians to have their patients undergo molecular testing in order to guide therapy decisions. Technologies emerging from the life sciences sector are now providing clinicians with richer data and information that will soon become part of the mainstream diagnostic algorithm. For example, clinical studies with the Translational Genomics Research Institute and U.S. Oncology aim to discover the most effective way to utilize DNA sequence data to guide cancer therapy decisions in triple-negative breast cancer patients. When fully harnessed, these technologies will have a profound impact on healthcare outcomes and the value that the laboratory provides to the clinician.—Frederick Eibl, Life Technologies (Carlsbad, CA); and Romain Prieur, formerly of Life Technologies

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