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Molecular Biology

QC & PT

Quality Assurance (QA) in the molecular laboratory is bifurcated into two distinct monitoring systems. Quality Control (QC) is the internal, daily process of running known standards alongside patient samples to verify that the assay worked today. Proficiency Testing (PT) is the external, periodic process of testing unknown samples sent by a regulatory agency to verify that the laboratory’s methods are accurate compared to the rest of the industry. Both are required for accreditation and patient safety

Assay Controls (Internal QC)

Because molecular amplification occurs in closed tubes and results are inferred from fluorescence or gel bands, the laboratory scientist cannot visually confirm that a reaction took place. Controls are the only mechanism to validate the results. A valid run requires a hierarchy of controls that monitor for contamination, amplification efficiency, and sample inhibition

  • The No Template Control (NTC)
    • Function: Monitors for Contamination. It contains all Master Mix reagents but uses water instead of nucleic acid
    • Interpretation: Must be Negative. If amplification occurs in the NTC, it indicates that “carryover” amplicons or environmental DNA have contaminated the reagents. The run is Invalid and must be repeated with fresh reagents
  • The Positive Control (PC)
    • Function: Monitors for Reaction Failure. It contains a known target sequence
    • Sensitivity Check: Ideally, a “Low Positive” (near the Limit of Detection) is used. If reagents degrade (e.g., enzyme loss), a strong positive might still work, but the low positive will fail, alerting the lab to a loss of sensitivity
    • Interpretation: Must be Positive within a specific Ct range. If the PC fails to amplify, the run is Invalid (likely due to thermal cycler failure or expired reagents)
  • The Internal Control (IC)
    • Function: Monitors for Inhibition and Extraction Efficiency within each specific patient tube. It targets a housekeeping gene (endogenous) or a spiked synthetic target (exogenous)
    • Critical Interpretation
      • Target (+) / IC (-): Valid Positive. (Strong viral targets can out-compete the IC)
      • Target (-) / IC (+): Valid Negative
      • Target (-) / IC (-): Invalid. The reaction failed completely, likely due to inhibitors (e.g., Heme, Heparin) in the patient sample. The result cannot be reported as Negative; it must be re-extracted or diluted
  • Quantitative Controls (Standard Curve)
    • For Viral Load assays, a set of standards is used to define the relationship between Ct value and Concentration
    • QA Metrics: The Slope (Efficiency) should be between -3.1 and -3.6. The Correlation Coefficient (\(R^2\)) must be \(>0.98\). Deviations indicate pipetting errors or reagent issues

Proficiency Testing (External Quality Assessment)

Proficiency Testing (PT) acts as a “final exam” for the laboratory. Three times a year, an approved agency (e.g., CAP, API) sends “blind” samples to the laboratory. The lab tests them and submits results for grading against peer laboratories

  • The “Treat as Patient” Rule
    • Regulatory Requirement: PT samples must be integrated into the routine workload and tested by the same personnel who run patient samples, using the same primary method
    • Prohibitions
      • Do not run PT samples in duplicate unless you run patients in duplicate
      • Do not assign PTs only to the Lead Tech or Supervisor
      • Inter-Laboratory Communication: It is strictly prohibited to compare results with another lab before the submission deadline
    • The “Fatal Flaw” (PT Referral): Sending a PT sample to a Reference Lab for confirmation/testing is defined as “Cheating.” This results in the automatic revocation of the lab’s CLIA certificate and a ban on testing
  • Grading & Performance
    • Peer Group: Results are compared to other labs using the exact same instrument and method to account for kit-specific biases
    • Quantitative Grading: Uses the Standard Deviation Index (SDI). A result is usually acceptable if it falls within \(\pm 2.0\) SD of the peer group mean
    • Qualitative Grading: Based on Consensus (typically 80% agreement among peers)
  • Performance Status
    • Satisfactory: Scoring \(\ge 80\%\) on an event
    • Unsuccessful Performance: Scoring \(< 80\%\) on 2 out of 3 consecutive events. This may require the laboratory to Cease Testing for that analyte until remediation is complete
  • Alternative Assessment
    • If no commercial PT kit exists for a rare analyte (e.g., a specific LDT), the lab must perform an alternative assessment twice a year. This is commonly done via Split-Sample Analysis, where a patient sample is tested in-house and then sent to a reference lab to confirm concordance