The Clinical, Analytical and Regulatory Issues Involved in Incorporating New Molecular Diagnostic Assays into the Clinical Lab

The function of molecular diagnostics is to analyze the composition of a patient’s genetic makeup in order to reveal any potential predispositions of that individual to specific diseases. Identifying these biomarkers can allow treatment options to be outlined that are likely to be effective in particular patients and not in others. Molecular diagnostic tests can work in several ways. They can quantify the levels of certain genetic materials that may be expressed by a bacterium, virus or cancer, or they can sequence specific regions of DNA to pinpoint genetic mutations.

By implementing the latest technology, laboratories can measure DNA, RNA and protein concentration, while also determining sample purity in accordance with A260/A280 and A260/A230 ratios.

The field of molecular diagnostics is being revolutionized through the development of biomarker identification and the continuous improvement of molecular techniques. However, translating these techniques from the bench to the clinic is an ongoing and delicate process. In particular, proteomic, genomic, and multiplex-based assays require extensive optimization before any results may be interpreted and used appropriately in a clinical setting. This article will discuss the clinical, analytical, and regulatory issues involved in the development and incorporation of new molecular diagnostic assays into the clinical laboratory.

Regulation and accreditation within the laboratory

Laboratory diagnostics, as the backbone of medical treatment, diagnosis and prevention, influence the majority of all hospital health care decisions. Attempts to enhance laboratory quality aim to reduce diagnostic errors and decrease turnaround time. It is also essential to ensure full traceability of all laboratory procedures to minimize risk and assure the safety of patients. This often requires a tailor-made approach for each individual laboratory, with the basic criteria being comprehensiveness, availability, response time, reliability, and accuracy of information.

Laboratory accreditation helps to ensure smooth quality control by providing means for third-party certification of the competence of laboratories to perform specific types of testing and calibration. Accreditation provides formal recognition of competent laboratories and, as a result, provides a ready means for customers to find reliable testing services in order to meet their demands.

In the United States, two of the key accreditation certifications for clinical laboratories are the Clinical Laboratory Improvement Amendments (CLIA) and the College of American Pathologists (CAP) accreditation programs. CLIA and CAP accreditation is regarded throughout the diagnostic industry worldwide as signifying that a laboratory performs diagnostic testing at the highest quality standards.

CLIA certification

The U.S. Congress passed CLIA in 1988, establishing 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 set standards for quality control (QC), 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.

However, because current CLIA regulations are not optimized for molecular diagnostic tests, clinical laboratories may choose to be certified through a professional organization such as the CAP accreditation program.

CAP accreditation

The CAP accreditation program helps clinical laboratories establish and maintain quality standards. The accreditation is based on standards that are categorized into specific checklists that provide detailed plans for laboratories to follow. An advantage of the CAP program is that it covers a variety of disciplines, providing an optimized checklist for molecular diagnostics and testing procedures.

To ensure that correct processes are put into place and maintained on a routine basis, it is vital that all laboratories identify the best accreditation program for their needs.

Authorizing new testing protocols

According to the CLIA, every 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. Method validation should be used to define the detection limits of the test and should also estimate the reproducibility, reliability, accuracy, sensitivity, specificity, and dynamic range of the test. Literature review, clinical trial data, current clinical practice, and regulatory guidelines are all used to assess clinical validity; platform description and instrument and software validation also play an essential role.

Analytical validation

A molecular diagnostic assay should be appropriately 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, test sensitivity and specificity, and the predictive value of a positive and negative test. The population of a validation study should be carefully selected. Consideration should be given to whether the results of a particular study will apply to individuals with a specific disease at varying stages and if the results can be extrapolated to a different population. Focus should also be on developing a sensitive and specific biomarker and not on achieving statistical significance. Clinical validity of a test cannot rely solely on the statistical differences found between affected and non-affected individuals participating in the validation studies. Sensitivity and specificity are key to successful translation of a test from the bench to the clinic.

Proteomic and gene expression patterns used as biomarkers require special statistics due to the possibility that genomic- and proteomic-based assays could cause overfitting of data. As a result, analysis should be carried out by a statistician with experience in working with scaled data sets.

Various factors can affect the robustness of a molecular assay, including tissue sampling and handling; tissue stability; and nucleic acid isolation, 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. Advanced spectrophotometers now enable the analysis of sample volumes as small as 0.5 - 2.0 μL with a dynamic range of 2 - 5,000 ng/μL for nucleic acids and without the need for a cuvette or dilutions. The spectrophotometers are able to determine nucleic acid concentration and generate full absorbance spectral data. The spectral data provided can offer analysts additional information regarding the presence of potential chemical contaminants such as phenol, glycogen, guanidine and Ethylenediaminetetraacetic, 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 and, as such, a molecular assay developed for diagnostic purposes should have a broad dynamic range and allow reliable detection of low levels of the biomarker detected by the assay.

Present landscape of 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 and 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, and miRNA arrays. These emerging molecular techniques are being developed for a wide range of applications, such as disease prediction, companion diagnostics, prognosis and characterization of unknown tumors, prediction of treatment efficacy, and personalized medicine.

There are numerous challenges 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 highquality 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, such as the Thermo Scientific NanoDrop 2000c, laboratories can measure DNA, RNA, and protein concentration, while also determining sample purity in accordance with A260/A280 and A260/A230 ratios. In addition, the instrumentation is preconfigured 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.

Improving patient experience

In the present biomedical landscape, the effective transition of instruments from the bench to the clinic is vital. Yet there are considerable challenges in achieving this transition smoothly and efficiently. Before results can be appropriately interpreted for clinical use, assays require extensive optimization to ensure accuracy and safety. Overcoming this challenge necessitates rigorous validation processes and QC procedures, along with technologically superior instrumentation. With these assurances in place, patients can be provided with improved screening, monitoring and treatment of various disease conditions.

Dr. Ilsa Gomez-Curet, Bioscience consultant, Thermo Scientific NanoDrop Products, can be reached at igomezcu@netscape. net or by phone at 302-479-7707.

For more information on the Thermo Scientific NanoDrop family of products, please visit www.thermoscientific.com/nanodrop or call 302- 479-7707.