In this article:
By Frederick L. Kiechle, M.D., Ph.D.
Editor's Note: Starting a molecular lab involves a lot of planning, especially in terms of space and design. How can you optimize your space and allow for the flexibility needed for future technology needs? What kind of services will you provide that will impact space design? Have you considered storage, safety issues and quality control? All of these issues come into play when developing a molecular lab. Here are some tips to get through them and make the right decisions.
In 1997, Rosalyn Yalow won the Nobel Prize for Physiology and Medicine for the groundbreaking laboratory method radioimmunoassay. This method was usually performed in the radioimmunoassay (RIA) lab — a specific space, in a centralized location. As radiolabeled techniques were replaced by non-radiolabeled methods, the RIA lab became the defacto owner of all immunoassays. This change lead to diseconomies of scale, including duplication of analytical equipment. Some of these immunoassays were performed in the centralized RIA lab and also in a decentralized special chemistry laboratory. 1
This same space issue exists for the molecular diagnostics lab; where will it be located? The centralized core lab for anatomic and clinical pathology applications; or a decentralized molecular lab located within a clinical or anatomic pathology lab, like microbiology for infectious disease identification? 2-4The final size of the molecular diagnostics clinical laboratory depends on the number of assays and associated techniques that you ultimately decide to offer to customers based on clinical need. The turnaround time for molecular identification of an infectious agent should be less than three to four days from a reference lab. This timeframe, and your service abilities, should help you select your first group of assays for this subset of infectious agents.
The layout of a 1990 molecular lab was in three distinct areas:
1. Sample preparation
3. Post-PCR location
This was driven by the fear of contamination during the three common steps in the molecular assay:
1. Nucleic acid extraction (preparation)
2. Amplification usually by PCR (pre-PCR)
3. Detection or quantization of amplified product (post-PCR) 2-4Kary B. Mullis won the Nobel Prize for Chemistry in 1993 for the development of PCR. The introduction of contamination safeguards, closed amplification instruments and fully automated systems has permitted many molecular techniques to be performed outside this traditional molecular laboratory.3
To reduce the risk of potential contamination, you should substitute dTTP with dUTP and remove dU incorporated into PCR-created DNA with the enzyme uracil DNA-glycosylase (UDG). The strands of DNA with dU will be destroyed after heating.4Work surfaces should be equipped with overhead ultraviolet lights, which are effective against DNA fragments greater than 300 bps.2 Corrosive-resistant countertops should be washed daily with 10 percent bleach solution (1 part regular bleach, nine parts water) and alcohol washes. 2,4 Each work area should have dedicated supplies and reagents, as well as lab coats and gloves.
In the pre-PCR area, master mixes are stored in airtight storage bags. You will need all reagents for PCR and positive/negative controls except for the input nucleic acid. The room should have a slight positive air pressure. In contrast, the post-PCR location has a slightly reduced pressure to pull air in from the outside and prevent the escape of the amplicons measured in the post-PCR space from escaping.In general, strict adherence to proper lab techniques will prevent contamination. Before determining the types of precautions that may be needed, always check carefully each step of the protocol to determine which steps are most likely to introduce contamination. Then develop a plan to minimize the occurrence of these events.
In the future, Laboratory Information Systems (LIS) functionality may be useful to increase workflow efficiency and tracking for surgical specimens during a molecular diagnostics evaluation. LIS focus on providing functionality for high-volume lab sections.For example, Gomah et al.5describe a project to define workflow in molecular oncology that used surgical specimens as their source of nucleic acid. The specimen size may range from a very small biopsy to a large surgical resection of the tumor. Each tumor type has a specific algorithm associated with it that may include tumor-specific test panels or sequential/reflex testing of a range of different genes, exons or particular codons. The end product included programming elements to reduce test complexity within a tool kit of features to facilitate sample handling from receiving to reporting. This example illustrates how LIS functionality may be useful to increase workflow efficiency and tracking for surgical specimens during a molecular diagnostics evaluation in the future lab.
The three-room or modified two-room concept of a molecular diagnostics lab design will probably be altered for future applications. This affected by workflow from a computer-assisted, simplified plan for specimen processing, amplification signal detection, and results interpretation.5The old concept of one specimen equals one assay will become one specimen equals multiple assays in the future. Molecular laboratory directors in the future will need to embrace the flexibility required to redesign the molecular laboratory to assure no specimen contamination occurs in an environment where multiple assays will be required for nucleic acid extracted from one specimen.
Initial space of a molecular lab should be assigned at 5,000 square feet or more to provide room for service-level expansion. This expansion will require more extractors and biological hoods in the clean areas and sequences, amplification and detection devices in open laboratory space. There are no inflexible guidelines in molecular lab design. The primary emphasis is avoidance of contamination and each step of the process workflow.
- Kiechle FL, Main RI. The Hitchhiker’s Guide to Improving Efficiency in the Clinical Laboratory. AACC Press: Washington DC. 2002: 40-41.
2. Dieffenbach CW, Dveksler GS. Setting up a PCR laboratory. Genome Res 1993;3:52-57.
3. Kiechle FL, Holland CA, Karcher R. Laboratory design. J Clin Ligand Assay 2005;28:186-197.
4. Mifflin TE. Setting up a PCR Laboratory. Cold Spring Harbor Protocols 2007:5-10; doi:10.1101/pdb.top14
5. Gomah ME, Turley JP, Lu H, Jones D. Modeling complex workflow in molecular diagnostics. Design specifications of laboratory software for support of personalized medicine. J Molecular Diagnostics. 2010;12:51-57.