Rachel Lee, Ph.D.

Texas Department of State Health Services

NBS Molecular Training Workshop

March 12, 2015
Quality Management Components
 Organization
 Personnel
 Documents and Records
 Advisory Services
 Laboratory Equipment
 Purchasing, Inventory, and Evaluation of Vendor Qualification
 Process Management
 Information Management
 Nonconforming Event Management
 Assessments
 Continual Improvement
 Facilities, Environment, and Safety
 Use of Referral Laboratories
 Laboratory Regulatory and
Accreditation Guidelines
 US Food and Drug Administration (FDA):
 Approves kits and reagents for use in clinical testing
 Proposed oversight for Laboratory Developed Test
 Clinical Laboratory Improvement Amendments (CLIA):
 Regulations passed by Congress 1988 to establish quality standards
for all laboratory testing to ensure the accuracy, reliability and
timeliness of patient test results regardless of where the test was
performed
 College of American Pathologists (CAP):
 Molecular Pathology checklist
 State Specific Regulations
 NY Clinical Laboratory Evaluation Program (CLEP)
Professional Guidelines
 American College of Medical Genetics (ACMG)
 Professional Guidelines
 Clinical and Laboratory Standards Institute (CLSI)
 Professional Guidelines
 Clinical and Laboratory Standards Institute (CLSI)
Contamination
 Introduction of unwanted nucleic acids into specimen
 the sensitivity of PCR techniques makes them vulnerable to
contamination

 Repeated amplification of the same target sequence leads

to accumulation of amplification products in the
laboratory environment
 A typical PCR generates as many as 109 copies of target sequence
 Aerosols from pipettes will contain as many as 106 amplification
products
 Buildup of aerosolized amplification products will contaminate
laboratory reagents, equipment, and ventilation systems
Potential Sources of Contamination

 Cross contamination between specimens

 Amplification product contamination
 Laboratory surfaces
 Ventilation ducts
 Reagents/supplies
 Hair, skin, saliva, and clothes of lab personnel
What happens if lack of contamination
control
 Incorrect results
 Require extensive cleanup
 Loss of creditability
 Impact on financial and performance
How to Control Contamination
 Laboratory design

 Laboratory practices

 Chemical and enzymatic controls

Setting Up a Molecular Laboratory
 Mechanical barriers to prevent contamination
 Spatial separation of pre- and post-amplification
work areas
 Area 1 – Reagent preparation
 Area 2 – Specimen/control preparation, PCR set-up
 Area 3 – Amplification/product detection, plasmid
preparation
 Physically separated and, preferably, at a substantial
distance from each other
 Unidirectional Flow
 Both personnel, including
cleaning personnel, and
specimens
 Amplification product-free to
product-rich
 Remove PPE before leaving
one area
 Avoid or limit reverse
direction
 Reusable supplies in the
reverse direction need to be
bleached.
CLSI MM19-A Establishing Molecular Testing in Clinical Laboratory
Environments
 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

 Dead air box with UV light – serves as a clean bench

area
Features of the 3 Areas
 Air pressure
 Reagent Prep – Positive
 Sample Prep - Negative
 Postamplification - Negative

 Reagent Prep – Single entrance, reagents used for

amplification should not be exposed to other areas
 Specimen Prep – Specimens should not be exposed
to post-amplification work areas
 Size of each area should consider space for
equipment and bench space needed for preparation
Laboratory Design Example 1
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.
Laboratory Design Example 2
http://www.roche-applied-science.com/campaigns/DeveloperTips/pcr/Physical-separation.html
Laboratory Design Example 3
http://fx.damasgate.com/the-pcr-laboratory/
Two Areas Only
 Area 1 – Reagent prep, specimen prep, and target
loading – use of laminar-flow hoods
 Area 2 – Amplification/product detection
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
Other Laboratory Design
Considerations
 Temperature and humidity requirements
 Exhaust ventilation
 Water quality
 Electric outlet
 Back-up power system
 Eye wash
 Ergonomic assessment
 Laboratory Practices
 Use of positive displacement pipettes and disposable
filtered pipette tips
 Avoid production of aerosols when pipetting
 Use of sterilized single-use plasticware
 Use of cleanroom sticky floor mats
 Minimizes the risk of amplicon carry-over on clothing,
hair and skin
 Hairnet
 Dedicated safety glasses
 Disposable labcoat/gown, color-coded preferred
 Gloves, need to change periodically
 Shoe covers
More Laboratory Practices
 Clean punches between samples
 Use of nuclease free or autoclaved water
 Aliquot oligonucleotides – multiple freeze thaws will
cause degradation
 Always include a blank (no template) control to
check for contamination
 Use of electronic data system (flow of paper)
 Wipe test (swab test)
 Monthly
 Detect, localize, and remove contamination
 Identify the source of the contamination
Decontamination Approaches
 Clean the work area & equipment routinely
 Clean the PCR workstation at the start and end of each
work day/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?
 Introduce a new testing system
 New analyte
 Analyte previously measured/detected on an alternate system
 An analyte added to a test system
 A modification to a test system
 Applies to
 Unmodified, FDA-cleared or approved method
 Modified, FDA-cleared or approved method
 In-house method
 Standardize method such as textbook procedure
 Determine 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 Calibrators
 Types of Controls
 Internal Control
 Internal positive amplification controls to detect failure of DNA
extraction or PCR amplification
 Reagent or equipment issues
 Integrity of DNA sample
 Presence of inhibitory substance

 External Control
 Positive control
 Negative control (normal, wild type)
 No template control (extraction blank)
 Blank
 Internal Controls
Tetra-primer ARMS-PCR

Simultaneous amplification of:

 Positive amplification control
 Mutation allele
 Reference allele

Alternative to tetra-primer ARMS is to

include an additional primer set to
amplify a different control sequence

Reference gene (e.g. RNaseP)

 External Controls
 Positive and negative controls:
 Inhibitors
 Component failure
 Interpretation of results
 Sources:
 Residual DBS
 PT samples
 QC materials
 No template controls and Blanks:
 Nucleic acid contamination during extraction
 Nucleic acid contamination during PCR
Frequency and Number of External
Controls
 Based on risk
 Ideally should represent each target allele and include in
each run, but may not be feasible when:
 Highly multiplex genotypes
 Systematic rotation of different alleles as positives
 Specimens representing short and long amplification products to control for
differential amplification
 Rare alleles
 Quantitative PCR
 External controls should represent more than one concentration, covering the
analytical measurement range
 Daily run or with each runs
 After equipment maintenance, new operator, new reagent
lot/shipment
Calibrators
 Calibrator copy levels should cover analytic cut-
offs
Evaluation of Controls and Calibrators
 Pass/Fail Criteria – established during validation study
 Parameters
 Specific PCR product bands
 Specific DNA fragments
 Quantity or Ct of reference gene
 Quantity or Ct of targeted marker
 Slope, R2, and Y-intercept of Calibrator curve
 Threshold
 Presence or absence of DNA bands
 Above or below LoB
 Above or below cut-offs
 Within Mean± 2SD, Mean ± 3SD, or Mean± 10%
 % of controls acceptable
 Impact the entire run or only affected samples
Allele drop-out (ADO)
 The failure of a molecular test to amplify or detect one or
more alleles
 Potential causes:
 DNA template concentration
 Incomplete cell lysis
 DNA degradation
 Non-optimized assay conditions
 Unknown polymorphisms in target sites
 Reagent component failure
 Interfering substance, http://www.aphl.org/aphlprograms/newborn-screening-and-
genetics/Pages/Assuring-Laboratory-Quality.aspx

 Major concern for screening laboratories

 Confirmation of mutation inheritance in families may not an option
 False Amplification
Potential causes:
 Non-optimized assay conditions

 Unknown polymorphisms in target sites

 Gene duplications
 Oligonucleotide mis-priming at related sequences
 Psuedogenes or gene families
 Oligonucleotide concentrations too high

 Nucleic acid cross-contamination

What to do if control fails?
Quality Indicator
Measurement to monitor and record specific activities as part of
the quality management system

 Turnaround Time

 % of failed runs

 Population medium

 Calibrator parameters

 Graph to identify trend or shift

 Monitor frequency and acceptable range

 Proficiency Testing
 Assessment of the Competence in Testing

 Required for all CLIA/CAP certified laboratories

 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

 CDC NSQAP  SeraCare

 UKNEQS  Corielle

 EuroGentest  ECACC

 CAP  In-house samples

 Maine Molecular  Round-robin with other NBS

laboratories
Sample Acceptance and Tracking
 Special specimen acceptance criteria?

 Assign a unique code to each patient

 Use two patient-identifiers at every step of the procedure

 Develop worksheets and document every step

 LIMS interface and Positive ID

Reagents
 Labeling Reagents:
 Content, quantity, concentration
 Lot #
 Storage requirements (temperature etc.)
 Expiration date
 Date of use/disposal
 Know your critical reagents (enzymes, probes, digestion
and electrophoresis buffers) and perform QC checks as
appropriate
 Critical Molecular Assay Components
 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
 Enzymes
 Benchtop coolers recommended
 Fluorescent reporters
 Limit exposure to light
 Amber storage tubes or wrap in shielding (foil)
Other QA/QC Considerations
 Specimen storage
 Laboratory Cleanliness, and Waste Disposal
 Instrument Maintenance and Calibration
 Instrument/Method Comparison
 Document Management
 Personnel Training and Competency
 Periodic Review of QA/QC
 COOP Plan
New CAP Requirement on TAT
 CBG.20140 Out-of-Range/Invalid Results Phase II
There is a policy for reporting positive (out of range) or invalid results to
the submitting location and other appropriate entities to allow for patient
follow-up within a timeframe appropriate to ensure maximum health
benefit.
NOTE: Positive results include those results that are outside of the expected range of testing results
established for a particular condition. Invalid results include situations where the laboratory is unable to
complete the screening process due to an unsuitable specimen, test, or incomplete information. The
findings must be communicated in a manner consistent with the urgency of the intervention needed. For
situations requiring repeat screening or confirmatory testing, the laboratory must clearly communicate the
timing of the actions to be taken.
Results must be reported to the submitting location (at minimum) within 7 days of specimen receipt and
within 3 days for specimens received for tests requiring additional action (e.g. invalid or positive).The
records should indicate when results were reported and who received the results. In cases where the testing
laboratory is responsible for documenting that a return specimen has been received and analyzed,
appropriate records should attest to specimen receipt, testing and result reporting.
Take Home Messages
 Separate laboratory spaces for Reagent Prep, Sample Prep,
and Amplification and Detection
 Precautions and special laboratory practices must be made
to minimize the risk of contamination
 A Quality Control Plan to monitor the quality of testing
process and detect errors should be in placed for each new
test before it’s implemented.
 Continuous quality improvement is essential