Molecular Laboratory Set up: Introduction, Design, Requirements, Laboratory Practices and Its Uses

Molecular Laboratory Design

Introduction of Molecular Laboratory

The molecular laboratory has become a growing part of the clinical laboratory. It includes all tests and methods to identify a disease and understand the predisposition for a disease analyzing nucleic acid i.e. DNA or RNA of an organism. Rapid advances in molecular diagnostics enable basic research and result in practical diagnostic tests which reflects the importance of molecular laboratory. The basic application is to determine changes in sequence or expression levels in crucial genes involved in disease. The use of molecular laboratory in molecular diagnostics, such as pre-implantation diagnostics or predictive genetic testing, still has technical problems as well as a novel, and to date unclear, social, ethical, and legal implications. The scope of molecular laboratory in molecular medicine could be expanded well beyond current nucleic acid testing which plays an important role in the practice of medicine, public health, pharmaceutical industry, forensics, and biological warfare, and drug discovery. The molecular laboratory marketplace offers a growth opportunity given the interest in utilizing molecular tools to precisely target therapeutics.

Design of Molecular Laboratory Setup

To design ‘Molecular Laboratory’ is really a hard task because of the following point of view-

  1. The laboratory should be compatible with mechanical barriers to prevent contamination which is the most common issue in the molecular laboratory.
  2. The spatial separation of pre-and post-amplification work areas
  3. Each area should be fascinated with adequate requirements.
  4. Unidirectional Flow
  5. Maintenance of air pressure
  6. Temperature and humidity requirements
  7.  Exhaust ventilation
  8.  Water quality
  9.  Electric outlet
  10. Back-up power system, etc.

The above design is modified on the following guidelines-

  • CLSI MM19-A Establishing Molecular Testing in Clinical Laboratory
    Environments
  • Mitchell P. S. et al. Nucleic Acid Amplification Methods: Laboratory Design and Operations, 2004, In
  • “Molecular Microbiology: Diagnostic Principles and Practice, edited by D. H. Persing et al” 99. 85-93.
  • http://www.roche-applied science.com/ campaigns/ DeveloperTips/ pcr/Physical-separation.html
  • http://fx.damasgate.com/the-pcr-laboratory/

Requirements of Molecular Laboratory 

Requirements of the molecular laboratory are as follows-

  1. Mechanical barriers to prevent contamination
  2. The spatial separation of pre-and post-amplification work areas
    Area 1: Sample Preparation Room
    Area 2: Reagent Preparation Room
    Area 3 – Amplification Room
  3.  Physically separated and, preferably, at a substantial distance from each other
  4. Unidirectional Flow
    √Both personnel, including cleaning personnel, and specimens
    √Amplification product-free to product-rich
    √Remove PPE before leaving one area
    √Avoid or limit the reverse direction
    √Reusable supplies in the reverse direction need to be bleached.
  5. Features of the 3 Areas
    √Each area has separate sets of equipment and supplies
    √Refrigerator/freezer (manual defrost)
    √ Pipettes, filtered tips, tubes, and racks
    √Centrifuge, timers, vortex
    √ Lab coat (color-coded), disposable gloves, safety glasses, and other PPE
    √Cleaning supplies
    √Office supplies
    √Ventilation system
  6.  Dead airbox with UV light – serves as a clean bench
    area
  7. Air pressure
    √Reagent Prep – Positive
    √Sample Prep – Negative
    √ Post amplification – Negative
  8. Reagent Prep – Single entrance, reagents used for amplification should not be exposed to other areas
  9. Specimen Prep – Specimens should not be exposed to post-amplification work areas
  10. The size of each area should consider space for equipment and bench space needed for preparation
  11. Alternative to Spatial Separation
    √Class II biological safety cabinet
    √ Dedicated areas for each work phase
    √Unidirectional
    √ Automated specimen processing station/closed tube amplification and detection system
  12. Other Laboratory Design Considerations
    √Temperature and humidity requirements
    √Exhaust ventilation
    √Water quality
    √ Electric outlet
    √Back-up power system
    √Eyewash
    √Ergonomic assessment

Laboratory Practices

Proper design and adequate requirements of the laboratory are not only sufficient without good laboratory practices and thus following points are useful for laboratory practices-

  1. Use of positive displacement pipettes and disposable filtered pipette tips
  2. Avoid production of aerosols when pipetting
  3. Use of sterilized single-use plasticware
  4. Use of cleanroom sticky floor mats
  5. Minimizes the risk of amplicon carry-over on clothing, hair, and skin
    √Hairnet
    √ Dedicated safety glasses
    √ Disposable lab coat/gown, color-coded preferred
    √Gloves, need to change periodically
    √ Shoe covers
  6.  Clean punches between samples
  7. Use of nuclease-free or autoclaved water
  8. Aliquot oligonucleotides – multiple freeze thaws will cause degradation
  9. Always include a blank (no template) control to check for contamination
  10. Use of electronic data system (flow of paper)
  11. Wipe test (swab test)
    √Monthly
    √Detect, localize, and remove contamination
    √ Identify the source of the contamination

Decontamination Approaches for Molecular Laboratory

 Clean the work area & equipment routinely
√Clean the PCR workstation at the start and end of each workday/run (UV light, 70% ethanol, fresh 10% sodium hypochlorite, DNA Away)
√ Clean the exterior and interior parts of the pipette
√Clean the equipment
√ Clean the doorknobs, handle of freezers

Chemical and Enzymatic Controls

Work stations should all be cleaned with 10% sodium hypochlorite solution (bleach), followed by removal of the bleach with ethanol and water.
Ultra-violet light irradiation-UV light induces thymidine dimers and other modifications that render nucleic acid inactive as a template for amplification
Enzymatic inactivation with uracil-N-glycosylase– Substitution of uracil (dUTP) for thymine (dTTP) during PCR amplification
√New PCR sample reactions pre-treated with Uracil-N-glycosylase (UNG) – contaminating PCR amplicons are degraded leaving only genomic DNA available for PCR

When is a Validation/Verification Study Required?

  1. Introduce a new testing system
    √ New analyte
    √Analyte previously measured/detected on an alternate system
  2. An analyte added to a test system
  3. A modification to a test system
  4. Applies to
    √ Unmodified, FDA-cleared, or approved method
    √Modified, FDA-cleared, or approved method
    √In-house method
    √Standardize methods such as textbook procedure
  5. Determine the analytic performance of an assay

Quality Control Plan

Monitor all steps of analytical procedure-

  • Types of Control
  • Frequency and Number of Controls
  •  Evaluation of Controls and Calibrator

False Amplification Potential causes

Potential cause-

  1. Non-optimized assay conditions
  2.  Unknown polymorphisms in target sites-√ Gene duplications
    √Oligonucleotide mispriming at related sequences
    √Pseudogenes or gene families
  3. Oligonucleotide concentrations too high
  4. Nucleic acid cross-contamination

Quality Indicator

Measurement to monitor and record specific activities as part of
the quality management system-

  1. Turnaround Time
  2. % of failed runs
  3.  Population medium
  4. Calibrator parameters
  5. The graph to identify trend or shift
  6. Monitor frequency and acceptable range

Proficiency Testing

  • Assessment of the Competence in Testing
  •  Performed twice a year
  •  If specimens are not commercially available alternative proficiency testing program has to be established (specimen exchange etc.)

Molecular Assay Proficiency Testing Material Sources

It may be of national or international origin.

Sample Acceptance and Tracking

  1.  Special specimen acceptance criteria?
  2. Assign a unique code to each patient
  3. Use two patient identifiers at every step of the procedure
  4. Develop worksheets and document every step
  5. Laboratory Information Management Systems (LIMS) interface and Positive ID

Reagents

  1.  Labeling Reagents: √ Content, quantity, concentration
    √ Lot #
    √ Storage requirements (temperature etc.)
    √ Expiration date
    √ Date of use/disposal
  2. Know your critical reagents (enzymes, probes, digestion, and electrophoresis buffers) and perform QC checks as appropriate.

Critical Molecular Assay Components

  1. Nucleic Acids: Prepare aliquots appropriate to workflow to limit freeze-thaw cycles
    √ Primers and probes
    √ dNTPs
    √ Genomic DNA
    √ 4-8°C
    √ -15 to -25°C
  2.  Enzymes-√  Benchtop coolers recommended
  3. Fluorescent reporters-√  Limit exposure to light
    √ Amber storage tubes or wrap in shielding (foil)

Other QA/QC Considerations

  1. Specimen storage
  2. Laboratory Cleanliness, and Waste Disposal
  3. Instrument Maintenance and Calibration
  4. Instrument/Method Comparison
  5.  Document Management
  6. Personnel Training and Competency
  7. Periodic Review of QA/QC
  8. COOP Plan: A COOP plan addresses emergencies from an all-hazards approach. A continuity of operations plan establishes policy and guidance ensuring that critical functions continue and that personnel and resources are relocated to an alternate facility in case of emergencies.

Uses of Molecular Laboratory

Uses of molecular laboratory in the field of clinical diagnostics as shown below-

  1. Health
  2. Medicine
  3. Forensics
  4. Pharmaceutical Industry
  5. Biological warfare
  6. Drug Discovery
  7. PCR based Technology
  8. Fluorescence in situ Hybridization (FISH)
  9. Biochip
  10. Peptide Nucleic acid (PNA)
  11. Proteomic Technology
  12. Electrochemical Detection of DNA

Key Notes

  1. Potential sources of contamination are cross-contamination between specimens, amplification product contamination, laboratory surfaces, ventilation ducts, reagents/supplies, and hair, skin, saliva, and clothes of laboratory personnel.
  2. Contamination may cause-
    √ Incorrect results
    √Require extensive cleanup
    √ Loss of creditability
    √Impact on financial and performance
  3. Contamination can be controlled using proper-
    √ Laboratory design
    √Laboratory practices
    √ Chemical and enzymatic controls

Further Readings

  1. https://www.aphl.org/programs/newborn_screening/Documents/2015_Molecular-Workshop/Molecular-Laboratory-Design-QAQC-Considerations.pdf
  2. https://www.researchgate.net/publication/303176803_Introduction_To_Molecular_Diagnostics
  3. https://www.scimmit.com/molecular-laboratory-design-and-its-contamination-safeguards/
  4. https://www.aacc.org/~/media/files/meetings-and-events/resources-from-past-events/conferences/2014/molecular-testing/may-29-and-30/checklist_for_mdx_testing_slides_may_29_2014.pdf?la=en
  5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2797718/
  6. https://cdn.intechopen.com/pdfs/23728/InTech-Good_clinical_laboratory_practice_gclp_for_molecular_based_tests_used_in_diagnostic_laboratories.pdf
  7. https://academic.oup.com/femspd/article/49/2/184/493227
  8. https://www.pomona.edu/academics/departments/molecular-biology/facilities
  9. https://www.bu.edu/emd/emergencyplanning/coop/
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