One of the goals of molecular diagnostics is the analysis of genetic content to reveal allelic variations that are important for a particular disease. These variations may affect an individual’s predisposition to develop a disease or the individual’s likelihood to respond to a particular treatment. By obtaining this information, patients are able to make informed lifestyle and treatment choices and physicians are empowered to select the optimal therapeutic strategy. Molecular diagnostic tests are most commonly used to identify genetic mutations or measure the expression levels of disease-associated genes that may be expressed by a bacterium, by a virus, or by cancer cells. Molecular diagnostics tests have also been successfully used to identify infectious diseases such as chlamydia, gonorrhea, and influenza and to diagnose and/or monitor conditions such as cystic fibrosis and cancer.
The development of new molecular techniques and the identification of new biomarkers are dramatically increasing the scope and value of molecular diagnostics. To fully leverage the benefit of these techniques, however, we must address the challenges associated with their translation from the bench to the clinic. Multiplex-, genomic-, and proteomic-based assays must be fully optimized to ensure accurate and appropriate interpretation of test results in a clinical setting. This article will explore the various aspects involved in the successful progression of new molecular diagnostics assays into the clinical laboratory, including current regulations and clinical and analytical considerations.
Laboratory regulations
As the backbone of screening, diagnosis, medical treatment, and prevention, molecular diagnostics influences many healthcare decisions. One of the goals of the clinical laboratory is to enhance service quality by reducing diagnostic errors and decreasing turnaround time. It is also essential for the clinical laboratory to ensure full traceability of all laboratory procedures to minimize risk and ensure the safety of all patients. Proper quality control (QC) and quality assurance (QA) measures in the molecular diagnostics laboratory often require the development of a tailor-made approach.
Laboratory accreditation by a third party ensures that smooth quality control measures are in place. In addition, such accreditation also ensures that the competence of laboratories to perform specific types of testing is itself tested and monitored on an ongoing basis. Accreditation provides formal recognition of competent laboratories and, as a result, provides customers with the means to find reliable testing services that meet their needs.
In the United States, two of the key accreditation programs for clinical laboratories are the Clinical Laboratory Improvement Amendments (CLIA) and the College of American Pathologists (CAP) programs. Accreditation by these programs is highly recognized in the molecular diagnostics industry worldwide, as only laboratories that have achieved and maintained the highest quality standards achieve this accreditation.
With regard to CLIA certification, the U.S. Congress passed the Clinical Laboratory Improvement Amendments (CLIA) in 1988. The CLIA program establishes quality standards for all clinical laboratories that run tests on human samples and provide results for the prevention, monitoring, diagnosis, or treatment of disease. CLIA has established standards for quality control, quality assurance, proficiency testing, and many other laboratory and administrative procedures. These standard practices ensure that lab tests are performed in an accurate, reliable, and timely manner. The requirements outlined by the CLIA regulations apply to clinical laboratories in all types of settings, including commercial, hospitals, medical centers, and physician offices.1
Because current CLIA regulations are not optimized for molecular diagnostic tests, clinical laboratories may choose to be certified through the accreditation program of a professional organization such as the College of American Pathologists (CAP).
The CAP accreditation program helps clinical laboratories to establish and maintain quality standards. The accreditation is based on standards that are categorized into specific checklists. The checklists provide a detailed plan for laboratories to follow. Advantages of the CAP program are that it covers a variety of disciplines and that it provides an optimized checklist for molecular diagnostics and testing procedures.2
It is vital that all clinical laboratories choose the best accreditation program for their needs; this ensures that the correct processes are put into place and maintained on a routine basis.
Validation of new molecular diagnostics assays
According to the CLIA, each laboratory is responsible for validating each new test before using it in a clinical setting. There are many elements involved in the validation of an in vitro diagnostic test, including its intended use and the environment in which it will be used. The analytical validation of a new method should define the detection limits of the test and should also estimate its reproducibility, reliability, accuracy, sensitivity, specificity, and dynamic range. Moreover, literature review, clinical trial data, current clinical practice, and regulatory guidelines are all used to assess clinical validity of a test. Validation of the instrumentation used to perform the assay and the software used to analyze the data also plays an essential role in the accreditation process.
A molecular diagnostic assay should be appropriately analytically validated before it can be used in a clinical setting. Numerous guidelines are recommended for successful analytical validation of a novel molecular assay. Goals must be clearly defined: namely, who the biomarker is for, and whether it is a primary test to evaluate disease risk or a secondary test to confirm disease. Analytical validation of a novel test should include estimation of critical parameters such as disease prevalence and test sensitivity, and specificity and the predictive value of a positive and a negative test.
The population of a validation study should be carefully selected. In addition, consideration should be given to whether the results of a particular validation study will apply to individuals with a specific disease at varying stages, or whether the results can be extrapolated to a different population. The focus of an analytical validation should be on developing a sensitive and specific biomarker and not on achieving statistical significance. Clinical validity of a test cannot solely rely on the statistical differences found between affected and non-affected individuals participating in the validation studies; sensitivity and specificity are key for successful translation of a test from the bench to the clinic.
Proteomic and gene expression patterns used as biomarkers require special statistics because of the potential of genomic- and proteomic-based assays to cause overfitting of the data. When this is the case, a statistician with experience working with scaled data sets should carry out the data analysis.
Various factors can affect robustness of a molecular assay, including tissue sampling and handling, tissue stability and nucleic acid isolation, sample preparation, concentration, and quality. Therefore, it is essential that effective QC measures for each molecular technique are used to minimize failure of downstream steps in the workflow. The latest microvolume quantification instrumentation, such as the Thermo Scientific NanoDrop 2000c UV-Vis spectrophotometer, can be implemented as a routine QC step to minimize consumption of precious samples and provide fast assessment of nucleic acid concentration and purity. The latest spectrophotometers enable the analysis of sample volumes as small as 0.5 – 2.0 uL, with a dynamic range of 2 -15,000 ng/uL for nucleic acids and without the need for a cuvette or dilutions. These spectrophotometers are able to determine nucleic acid concentration and generate full absorbance spectral data. The spectral data provided can offer additional information regarding the presence of potential chemical contaminants such as phenol, glycogen, guanidine, and ethylenediaminetetraacetic (EDTA) that may be introduced by extraction procedures and have the potential to inhibit downstream applications.
Developing a robust and reproducible assay is as important as finding the biomarker; thus, a molecular assay developed for diagnostic purposes should have a broad dynamic range and should allow reliable detection of low levels of the biomarker detected by the assay.
Emerging methods in molecular diagnostics
Tests in molecular diagnostics investigate genes, metabolic pathways, drug metabolism, and disease risk or progression. Genetic tests focus on DNA and RNA sequences and how they are related to disease, while proteomic tests focus on the function, structure and chemical modifications of proteins and how these relate to the onset or progression of disease. In addition, the emerging category of metabolomic testing evaluates chemicals or metabolites such as lipids and carbohydrates.
Over recent years, rapid, sensitive, high-throughput methods have been developed with the ability to detect nucleic acid and protein variations on a genome-wide scale. Some of the emerging molecular techniques making their way into molecular diagnostics include microarrays, multiplex nucleic acid amplification techniques, mass spectrometry, high-density microarrays, next-generation sequencing, comparative genomic hybridization (CGH), and miRNA arrays. These emerging techniques are being developed for a wide range of applications including disease prediction, companion diagnostics, prognosis and characterization of unknown tumors, prediction of treatment efficacy, and personalized medicine.
Numerous challenges are associated with emerging molecular diagnostics. Clinical laboratories will need to know how to work with new and complex platforms (e.g., microarrays and next-generation sequencing) and how to store, analyze and integrate complex data from various sources. Also, close attention to sample extraction and processing is essential to ensure that high-quality starting material is used for downstream applications. When performing nucleic acid-based assays, variations in the quantity, purity and integrity of DNA or RNA samples can result in variable results and erroneous conclusions. By implementing the latest technology, clinical laboratories can measure DNA, RNA, and protein concentration, and determine sample purity by measuring the A260/A280 and A260/A230 ratios. In addition, the cutting-edge instrumentation is pre-configured to simplify and accelerate common applications for nucleic acid, microarray and protein quantification.
The implementation of proper QC measures is necessary for appropriate validation of emerging molecular techniques. These measures can identify the need for improvements in sample processing, workflow, or downstream processes and will ultimately aid in the development of a robust molecular assay that improves patient care and has minimal test-associated risks.
Bridging the gap
Bridging the gap between clinical research and patient care is an important goal in the current biomedical environment. The successful translation of clinical information will enable physicians to optimize screening, monitoring, and treatment of a patient. Significant challenges are associated with a successful transition of emerging molecular assays from the bench to the clinic. The current regulatory landscape stipulates rigorous optimization and validation of new molecular diagnostic assays. Proper assay validation and instrumentation ensure that laboratories accurately and appropriately interpret data for clinical use and that patients are provided with high-quality healthcare.
Ilsa Gomez-Curet, PhD, is a bioscience consultant for Thermo Scientific NanoDrop products and has more than 15 years experience working in biomedical research in academic, hospital, and industrial environments. For more information on the Thermo Scientific NanoDrop family of products, please visit www.thermoscientific.com/nanodrop or call +1 302-479-7707.
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