INFECTION DIAGNOSTICS :: CROSS-CONTAMINATION


This helps to ensure reduce the risk of contamination by keeping clean areas free of amplicons.


Pass through air locks. Use of pass- through cabinets can facilitate a unidirec- tional workflow. These boxes are designed to maintain complete environmental sepa- ration between work areas and minimize cross contamination. Dedicated Personnel.5


Laboratory


personnel dedicated to either clean room activities or dirty room activities is recom- mended. Clean lab coats and gloves should be worn when performing PCR; gloves should be changed frequently. In the event that laboratory personnel need to move from a dirty room back to a clean room, all personal protective equipment (PPE) should be changed, and hands washed. Dedicated consumables and equipment.5


The availability of dedicated storage (e.g., freezer, fridge) in each room prevents move- ment of reagents and samples between dedicated areas, thereby minimizing the potential for contamination. In addition, consumables (e.g., lab coats, gloves, goggles, disposable shoe covers, etc.) and equip- ment (e.g., pipettes, centrifuges, pipette tips, etc.) should never be brought from dirty rooms back into clean rooms. Aerosol-resistant pipettes. Sample to sample contamination can occur due to aerosols. Use of barrier tips prevents liquids and aerosols from contacting the pipette. Pipetting techniques. Use of proper


pipetting technique can also prevent contamination. This includes ensuring the pipette tip is securely seated on the pipette and aspirating at a vertical 90° angle with consistent smooth speed and pressure. When dispensing liquid, the pipette should be held at a 45° angle with the tip of the pipette touching the side of the receptacle to minimize splashing. No template control (NTC).7


NTCs


consist of all reagents added during prepa- ration of the PCR reaction mixture but uses water instead of the extracted sample. Including an NTC in PCR amplification reactions allows for the identification of contamination in samples, reagents, and/ or the lab environment.


Cleaning/decontamination techniques Surface and Equipment.8


Following ap-


propriate cleaning and decontamination techniques both before and after PCR reduces the potential for contamination of the PCR reaction. Frequent cleaning of work areas with freshly made 10-15% sodium hypochlorite solution (bleach) followed by rinsing with 70% ethanol or cleaning with commercially avail-


42 MAY 2022 MLO-ONLINE.COM


able DNA-destroying decontamination products is recommended. Alternatively, cleaning of work surfaces and equipment with 70% ethanol followed by irradiation with UV light can also be done; irradiation is critical as ethanol only precipitates but does not remove DNA. UV irradiation.9


This technique


induces thymidine dimer formation in the DNA rendering it unusable as a template for further amplification. Use of UV irradiation is recommended for laminar flow hoods for at least 30 minutes prior to use, followed by clean- ing with 70% ethanol.


Additional Considerations Quality Monitoring Statistics10


As men-


tioned, the greatest risk source of con- tamination for PCR-based tests is from exogenous DNA. Laboratories must have clearly written procedures in place to monitor for the presence of false-positive results. This can include implementa- tion of wipe or swipe tests on a defined schedule, positivity rate monitoring by reviewing summary statistics at the local and regional level, and investigations of physician inquiries. Wipe or swipe tests involve dampen- ing of a sterile swab in sterile saline and wiping potentially contaminated surfaces of a laboratory (e.g., benches, pipettes, handles of fridge/freezer, centrifuges, etc.). These swabs are then processed and tested in manner similar to patient samples. A positive result from a wipe test is indicative of environmental contamination. Contamination from Serology Testing11 In some scenarios, diagnosis of an infec- tious disease such as Hepatitis C (HCV) or Human Immunodeficiency Virus (HIV) begins by screening for anti-HCV or anti- HIV antibodies. If antibodies are detected in a sample, further testing a PCR-based method is required for confirmation. When the same sample used for serology testing is also used for nucleic acid testing the potential exists for increased risk of cross-contamination, dependent on the design of the laboratory instruments used for serology testing. For example, instruments using a washable sample probe pose a slightly increased risk for cross-contamination versus instruments that utilize a disposable tip. As a mitigation, it has been suggested that low level RNA results from samples previously tested on a serology instrument with a washable probe be rejected and a new sample requested for repeat PCR analysis. Alternatively for samples screened on an instrument using disposable tips, there may be no need for a separate tube for confirmatory PCR testing.


Conclusion The safeguards and operational recom- mendations summarized above serve to prevent contamination of molecular diagnostic tests and subsequent impact to diagnostic and treatment decisions. In many scenarios,molecular testing plays a major role in the management of patients, thus prevention of contamination must be a priority in clinical molecular laboratories.


REFERENCES


1. Aslanzaden J. Preventing PCR amplification carryover contamination in a clinical laboratory. Ann Clin Lab Sci. 2004;34(4):389-396.


2. Hu Y. Regulatory concern of polymerase chain reaction (PCR) carryover contamination. In: Samadikuchaksaraei, A, ed. Polymerase Chain Reaction for Biomedical Applications. IntechOpen, 2016:57-68. DOI: 10.5772/66294.


3. Huggett J, Benes V, Bustin S, et al. Caution- ary note on contamination of reagents used for molecular detection of SARS-CoV-2. Clin Chem. 2020;66(11):1369-1372. DOI: 10.1093/clinchem/ hvaa214.


4. Mifflin TE. Setting up a PCR laboratory. CSH Protoc. 2007; pdb.top14.


5. Standards Unit, National Health Service, Public Health England. UK Standards for Micro- biology Investigations: Good practice when performing molecular amplification assays. Updated February 19, 2018. Accessed March 20, 2022. https://assets.publishing.service.gov. uk/government/uploads/system/uploads/attach- ment_data/file/682533/Q4i5.pdf.


6. Aysal A, Pehlivanoğlu B, Ekmekci S, Gündoğdu B. How to set up a molecular pathology lab: a guide for pathologists. Turk Patoloji Derg. 2020;36(3):179-187. DOI: 10.5146/ tjpath.2020.01488.


7. Viana RV, Wallis CL. Good Clinical Laboratory Practice (GCLP) for Molecular Based Tests Used in Diagnostic Laboratories. In: Akyar I, ed. Wide Spectra of Quality Control. InTech, 2011: 29-52.


8. Prince AM, Andrus L. PCR: how to kill unwanted DNA. Biotechniques. 1992;23(3):358-360.


9. Ou CY, Moore JL, Schochetman G. Use of UV irradiation to reduce false positivity in polymerase chain reaction. Biotechniques. 1991;10:442-446.


10.Wallace P, McCulloch E. Quality Assurance in the Clinical Virology Laboratory. Encyclo- pedia of Virology. 2021;64-81. DOI:10.1016/ B978-0-12-814515-9.00132-6.


11. Rondahl E, Gruber M, Joelsson S, et al. The risk of HCV contamination in serology screen- ing instruments with a fixed needle for sample transfer. J Clin Virol. 2014;60:172-173.


Jeanne Rhea-McManus, PhD, MBA, DABCC, NRCC has been with Sie- mens Healthineers for 7 years, previously as a Medical Officer and currently as the Senior Director of Medical Science Information and Communication.


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