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Molecular Biology

Quality Assurance

Quality Assurance (QA) in the molecular biology laboratory is a comprehensive management system designed to guarantee the integrity of the testing process. Unlike Quality Control (QC), which focuses on a specific run, QA encompasses the entire workflow, from the moment a specimen is collected to the moment the result reaches the clinician. The following overview details the critical control points required to maintain accreditation and ensure patient safety

Specimen Processing, Preparation, Transport, & Storage

The pre-analytical phase is the source of the majority of laboratory errors. Molecular assays are uniquely sensitive to enzymatic inhibition and nucleic acid degradation, requiring strict adherence to acceptance criteria

  • Collection & Transport: The choice of anticoagulant is paramount. EDTA (Lavender) is preferred as it prevents DNA degradation by chelating magnesium. Heparin (Green) is strictly prohibited as it inhibits Taq polymerase. RNA specimens require immediate processing or freezing (-70°C) to prevent degradation by RNases released from lysing white blood cells
  • Nucleic Acid Evaluation: Before amplification, the extracted material must be assessed
    • Quantity (Yield): Measured via UV Spectrophotometry (A260) for high concentrations or Fluorometry (e.g., Qubit) for high sensitivity
    • Quality (Purity & Integrity): Purity is assessed via absorbance ratios (A260/A280 ~1.8 for DNA, ~2.0 for RNA). Integrity is visualized via gel electrophoresis or microfluidic RIN scores to ensure the genetic material is not sheared or degraded

Reagent Selection, Preparation, & Documentation

Reagents are the chemical foundation of the assay. Because molecular reagents include biological enzymes and unstable probes, they require rigorous inventory management

  • Selection & Storage: Reagents must be “Molecular Grade” (Nuclease-Free). To prevent degradation from repeated freeze-thaw cycles, bulk reagents must be aliquotted into single-use volumes immediately upon receipt. Enzymes are stored at -20°C, while photosensitive probes must be protected from light
  • Preparation & Calculations: Master Mix preparation requires the use of the \(N+1\) Rule (calculating for the number of samples plus controls plus an overage) to account for pipetting dead volume. Dilutions follow the \(C_1V_1 = C_2V_2\) formula
  • Documentation: Complete traceability is required. All reagent logs must track Lot Numbers, Expiration Dates, and Date Opened. Reagents prepared in-house must be labeled with the preparer’s initials and the new expiration date

Assay Performance & Validation

Before a test can be used for patient care, the laboratory must prove that it works as intended. The regulatory pathway depends on the test type

  • Verification (FDA-Cleared): Verifying that the lab can reproduce the manufacturer’s claims regarding Accuracy, Precision, and Reportable Range
  • Validation (LDTs): A more extensive study required for Laboratory Developed Tests. This establishes the performance specifications from scratch, including:
    • Analytical Sensitivity (LoD): The lowest concentration detected 95% of the time
    • Analytical Specificity: Freedom from cross-reactivity with related pathogens
    • Linearity (AMR): The range over which the assay is accurate without dilution

Assay Troubleshooting

When an assay fails, a systematic approach is used to identify the root cause, distinguishing between reaction failure and contamination

  • False Negatives: If the Patient and the Internal Control both fail, Inhibition is the likely cause (matrix effect). If the Positive Control also fails, Reagent Failure or Thermal Cycler Failure is indicated
  • False Positives: If the No Template Control (NTC) amplifies, Contamination (carryover) has occurred. The run is invalid, and a deep cleaning/reagent replacement is required
  • Troubleshooting Logic: The corrective action path is determined by the specific combination of Control failures (NTC, Positive Control, Internal Control)

Result Calculation, Interpretation, & Reporting

The translation of raw data (fluorescence/Ct values) into a clinical report requires statistical analysis and standardized nomenclature

  • Calculation: Quantitative results are derived from a Standard Curve (Ct vs. Log Concentration). The reaction efficiency (slope) and correlation coefficient (\(R^2\)) of the curve must meet QA limits for the results to be valid
  • Interpretation: Results are interpreted in the context of the Internal Control. A negative result is only valid if the Internal Control amplifies. “Gray zone” results (late Ct values) require scrutiny of the amplification curve shape to rule out artifact
  • Reporting: Reports must use standardized units (IU/mL or Copies/mL) and unambiguous language (“Detected” vs. “Not Detected”). Required elements include the methodology used, the Limit of Detection, and specific regulatory disclaimers for LDTs

Quality Control (QC) & Proficiency Testing (PT)

This area covers the daily and periodic monitoring of laboratory accuracy

  • Internal QC (Daily): Must be included in every run
    • NTC: Monitors contamination
    • Positive Control: Monitors reagent and instrument function
    • Internal Control: Monitors extraction and inhibition in the individual patient tube
  • Proficiency Testing (Periodic): An external check where “blind” samples are sent by an agency (e.g., CAP). These must be tested exactly like patient samples (“Treat as Patient”) to verify accuracy against a peer group. Failure to pass PT can result in the suspension of testing privileges

Equipment & Instrumentation

Reliable data requires reliable hardware. Equipment QA involves a lifecycle of Validation, Calibration, and Maintenance

  • Thermal Cyclers: Must undergo temperature verification (accuracy and uniformity) and optical calibration (dye spectra) to ensure precise cycling and detection
  • Pipettes: The primary source of volumetric accuracy. They are calibrated semi-annually (Gravimetric analysis) to ensure precision. Maintenance involves checking O-rings to prevent leaking
  • Safety Equipment: Biological Safety Cabinets (Hoods) must be certified annually to ensure airflow protects the user and the sample. UV lights used for decontamination must be checked for intensity
  • Validation: All new equipment must undergo Installation Qualification (IQ), Operational Qualification (OQ), and Performance Qualification (PQ) before routine use