Quality Assurance, Quality Control, Safety, and Laboratory Operations
Quality control
Each run needs positive and negative controls, a no-template control, and an internal control to monitor extraction and amplification inhibition (endogenous genes such as RNase P, or exogenous armored RNA). Results are charted on Levey-Jennings plots and judged by Westgard rules: 1-3s and 2-2s are rejection rules, R-4s signals random error, and 4-1s or 10x indicate systematic shift. Investigate shifts, trends, and drift rather than repeating until acceptable.
Validation and verification
Validation establishes performance characteristics for a laboratory-developed test; verification confirms the manufacturer's claims before clinical use. Parameters include accuracy, precision, analytical sensitivity and specificity, reportable range, reference interval, and limit of detection. Proficiency testing, alternative performance assessment, competency documentation, calibration, temperature monitoring, and turnaround-time monitoring complete the quality program. Under CLIA, laboratory-developed tests require documented validation.
Contamination control
Use unidirectional workflow: extraction, then amplification setup, then detection, in separate areas with dedicated equipment and aliquoted reagents. Amplicon contamination is prevented with dUTP/UNG, fresh bleach (about 10%), and UV where appropriate.
Safety
Most molecular work is BSL-2 with standard precautions and PPE; culture of Mycobacterium tuberculosis and certain agents requires BSL-3. Know chemical hazards: phenol causes burns, guanidine thiocyanate plus bleach releases toxic gas, ethidium bromide is a mutagen, and formaldehyde is a carcinogen. Use SDS, spill kits, and compliant waste streams.
Traps
Common errors include believing 1-3s is only a warning, swapping validation and verification, and carrying amplified product into the clean extraction area.
Worked example
A single control exceeding ±3 SD triggers the 1-3s rule: reject the run, investigate, and repeat before reporting.