Molecular Diagnostics in the Medical Laboratory in Real Time

July 7, 2021

Molecular diagnostics (MDx) is the rapidly developing area of laboratory medicine that investigates human, viral and microbial genomes and the products they encode. Molecular diagnostic techniques and platforms are playing a larger and more critical role in all areas of anatomic and clinical pathology. In the last decade or so, the clinical laboratory has seen an explosion in the available menu of tests based upon DNA and RNA analysis. The completion of the Human Genome Project and the rapid advancement of technology to arise out of that effort has moved from the research bench to the clinical laboratory bench with swift success. For the first time in the history of the diagnostic laboratory, molecular pathology and diagnostics are extending the range of information available to physicians, pharmacists, geneticists, forensic scientists, research scientists and other healthcare professionals.

Historical and Current Examples of Molecular Diagnostics

While the ongoing SARS-CoV-2 / COVID-19 pandemic helped make molecular terms like ‘polymerase chain reaction’ (PCR), ‘false positive’ and ‘variant’ common, the field actually dates to 1949 with Linus Pauling and colleagues’ characterization of sickle cell anemia as a ‘molecular disease.’ However, it took decades for the scientific discipline of molecular biology to develop and become usable in the medical laboratory as a basis for disease diagnostics. MDx grew from the early days of recombinant DNA technology. Sequencing and cDNA cloning were critical for establishing basic knowledge on the primary sequence of various genes. DNA probes incorporating radioactive nucleotides allowed the analysis, via Southern blotting, of genomic regions, leading to the concept and application of restriction fragment length polymorphism (RFLP) to track variant alleles in the human genome. In 1976, Kan et al. were the first to make a prenatal diagnosis of α-thalassemia using MDx techniques. This diagnostic, alongside the use of RFLP to characterize sickle cell alleles, set the foundation for characterization of other genetic diseases (e.g., cystic fibrosis), as well as infectious diseases, using MDx platforms.
The development of PCR in the mid 1980s led to the golden era of molecular biology and MDx, and the use of a thermostable DNA polymerase from Thermus aquaticus (i.e., Taq polymerase, Saiki et al., 1988) quickly ushered this technique into the realm of laboratory medicine. With its powerful ability to exponentially amplify a target sequence, PCR allows the identification of a known mutation or sequence within hours. Not only did PCR bolster MDx in the clinical laboratory, it provided a foundation for the design and development of many variant detection schemes based on the amplification of DNA. It helped establish 3 categories for variant detection, depending on the basis for discriminating the allelic variants: (1) enzymatic-based methods (e.g., RFLP and oligonucleotide ligation assay), (2) electrophoretic-based methods (e.g., single-strand conformation polymorphism (SSCP), heteroduplex analyses (HAD) and denaturing gradient gel electrophoresis) and (3) solid phase-based methods (e.g., reverse dot-blot and allele-specific hybridization). While many of these methods are now infrequently used in clinical microbiology, they paved the way to current, more sophisticated methods.

Robin Forbes performs a PCR assay to detect a methicillin-resistant Staphylococcus aureus (MRSA) gene as Dr. Rodney Rohde observes.
Robin Forbes performs a PCR assay to detect a methicillin-resistant Staphylococcus aureus (MRSA) gene as Dr. Rodney Rohde observes in the Bachelor of Clinical Laboratory Science Program, a NAACLS-accredited MLS 2-year undergraduate program at Texas State University.
Source: Rodney Rohde.

After publication of the human genome draft sequence, the challenge to improve existing variant detection technologies to achieve robust, cost-effective, rapid and high-throughput analysis of genomic variation moved to the forefront of MDx. Important and critical advances continued in the world of MDx with the invention of real-time PCR and its numerous variations, DNA microarray-based genotyping and transcription profiling, microbiome sequencing (gut and other areas of the body), proteomics (detection of disease-specific protein profiles), pharmacogenomics, nutrigenomics, forensic medicine and CRISPR/Cas9 genomic editing.
Even with the advent and explosion of diverse variant detection assays, DNA sequencing is the gold standard for identification and surveillance of pathogens. This is especially true with breakthroughs in next-generation sequencing (NGS) technology. However, the costs for the initial investment and the difficulties in standardization and interpretation of ambiguous results continue to place limitations on the use of NGS in clinical laboratories. Physicians and other health care professionals are now working with MDx credentialed professionals to understand the basis of infectious disease pathology and when to use molecular diagnostics like NGS. One example is the use of 16S in-house assay sequencing to identify bacterial pathogens directly from tissue specimens when culture results are negative, but there is evidence of histopathologic pathogen damage.

Challenges in Molecular Diagnostics

Twenty-six years ago, the U.S. Food and Drug Administration approved the first direct-specimen molecular testing for infectious diseases. Since that time, the rapid advancement of molecular technology has been driven by 2 primary areas: (1) automated extraction, amplification and detection platforms and (2) next-generation sequencing. As with any new advanced area, there are challenges and limitations that the laboratory medicine and public health fields must pay close attention to as these developments intersect with the care of patients and healthcare and public health policy.
The cost of this advanced technology could lead to further health disparities if economic decisions limit the use of MDx to certain communities or populations. Another challenge of advanced and rapidly implemented MDx platforms is potential over- or underutilization. For example, rapid MDx platforms are often faster and more sensitive than traditional culture methods. However, the adoption of these MDx assays has been so quick that in some cases it outpaced evidence of clinical utility. The need for healthcare professional education (e.g., physicians, clinical pharmacy) is also a challenge to consider. Physicians must understand the limitations to and appropriate utilization of these technologies in order to provide cost-effective and well-informed care for their patients. 

The Medical Laboratory Needs MDx Professionals

The ability of a medical laboratory professional (or other appropriate laboratory professional) to perform molecular diagnostic testing has become critical to the laboratory medicine profession. Knowledge of methodology associated with detection and surveillance of pathogens, cancer biomarkers, inherited genetic disorders or other biomarkers is imperative for the current and future professional. The field is currently in need of well-trained medical laboratory professionals with strong biomedical science and medical laboratory science backgrounds and a thorough understanding of technologies used in assay development who can bridge the current state of practice with continuing developments in high complexity testing.
Students run Real-Time PCR assays in the Master of Science in Molecular Pathology laboratory at Texas Tech University Health Sciences Center.
Students run Real-Time PCR assays to detect infectious diseases in the Master of Science in Molecular Pathology laboratory, a NAACLS accredited DMS 1-year graduate program at Texas Tech University Health Sciences Center.
Source: Ericka Hendrix.
The best way to work in the field of MDx, command a handsome salary and learn to validate new molecular assays is to become a certified technologist in molecular biology. The American Society for Clinical Pathology (ASCP) Board of Certification (BOC) offers a certification exam for eligible candidates. A bachelor's or master’s degree from a National Accrediting Agency for Clinical Laboratory Sciences (NAACLS)-accredited Diagnostic Molecular Science (DMS) program or Medical Laboratory Science (MLS) program is the fastest route to become eligible to sit for the exam. There are currently 8 NAACLS-accredited DMS programs offering a variety of options, ranging from certificates to undergraduate and graduate degrees. Although less applicable to those interested in infectious disease, there are also NAACL- accredited program in cytogenetics, and ASCP (BOC) offers a technologist in cytogenetics (CG) certification exam. Currently, there are only 4 NAACLS-accredited programs in the U.S. for cytogenetics. These degrees (DMS and CG) give graduates the skills to immediately start working in the field and validating new molecular assays to expand the molecular testing menu for more personalized patient care.

The issues surrounding the advancement of molecular diagnostics will continue to grow in the race to enhance care for individuals using genomic and metagenomic information. Those in the field need to be adaptable, analytical and ethically responsible to forge a new and exciting path of personalized medicine.

Author: Ericka C. Hendrix, Ph.D.

Ericka C. Hendrix, Ph.D.
Ericka C. Hendrix, Ph.D., is an associate professor and director of the Master of Science in Molecular Pathology program at Texas Tech University Health Sciences Center.

Author: Rodney Rohde, Ph.D., SM(ASCP), SVCM, MBCM, FACSc

Rodney Rohde, Ph.D., SM(ASCP), SVCM, MBCM, FACSc
Rodney Rohde, Ph.D., is the Associate Director of the Translational Health Research Initiative at Texas State University.