In the previous post, we discussed some critical aspects of the pre-analytic phase of testing (Pre-analytic phase). To continue the series on the importance of diagnostic testing in light of the current COVID-19 (SARS-CoV-2) pandemic, we will discuss key aspects of the analytic phase of diagnostic testing in your clinical laboratory. In this post, I will consider some often-overlooked aspects of the analytic phase of diagnostic testing that pose high risks to patient safety and to the overall quality of the testing process.
The Analytic Phase
A lot of my recent and current work (Therapeutic Areas and Technologies) in building clinical diagnostic laboratories is with molecular assays that rely on an amplification step as part of the analytic phase of testing (e.g. COVID-19 RT-PCR assays, next generation sequencing to detect driver mutations in oncology, microarrays to detect gene expression, etc). These amplification-based assays have some unique requirements that are often neglected as assays and labs are established or as specimen volume increases. In this post, I will discuss three essential aspects of the analytic phase of testing for amplification assays that your lab needs to pay close attention to in order to ensure high quality testing and patient safety, even as labs are running at capacity.
Uni-directional Workflow & Decontamination Procedures
CAP/CLIA laboratories performing molecular assays need to pay special attention to a critical aspect of laboratory infrastructure—containment. Molecular assays with an amplification step have a risk of false positive results due to the risk of cross-contamination from amplicon in your facility from previous batches of samples. The best way to reduce the risk of cross-contamination is to ensure that the pre-amplification steps are physically separated from the post-amplification steps, using containment devices (e.g. separate rooms, PCR workstation, etc). A uni-directional workflow must be established within your facility so that the pre-amplification area is never re-entered by personnel, equipment, PPE, etc that have been exposed to the post-amplification portion of the workflow. Even with the proper equipment and procedures, the success of the uni-directional workflow requires a great deal of caution and discipline from your laboratory personnel in order to minimize carry-over and cross-contamination in the molecular diagnostic laboratory.
Despite having the most careful laboratory staff and the most contained environment, accidents and mistakes happen. To reduce your risk of false positive results due to contamination events, your clinical laboratory must have implemented procedures for environmental decontamination and monitoring that your laboratory staff adheres to regularly. Surfaces and equipment (where possible) should be wiped down with 10% freshly prepared bleach (wait 15 minutes), then rinsed with de-ionized water, followed by cleansing with 70% ethanol to ensure decontamination. Frequent wipe tests must be performed to ensure environmental contamination is minimal.
Instrument Maintenance and Calibration
Another way to ensure that the analytic phase of testing is robust is to maintain and calibrate all of your laboratory’s instrumentation according to the manufacturer’s recommendations. This one can slip through the cracks easily when the lab is running at capacity and everyone is stretched. On the flip side, consistent maintenance and calibration of your laboratory’s instrumentation is relatively easy and fast to perform and that small investment brings considerable payout by reducing the amount of systematic error in your laboratory’s results. For healthcare providers and patients served by your laboratory, your laboratory’s disciplined commitment to instrument calibration means high quality test results.
Quality Control: Assay Controls
For molecular assays, it is very important to include the proper controls for proper interpretation of results. In general, each batch of specimens should include an internal positive control that monitors a target and serves as a control for all processes and reagents in the assay (e.g. nucleic acid extraction, amplification, etc). The internal positive control can be either an endogenous target or an external target added to the specimen prior to nucleic acid extraction. If the internal positive control associated with a batch of specimens is negative, then the laboratory team needs to trouble-shoot the reagents and processes in order to understand what went wrong and make corrections. An external positive control should be included as part of your diagnostic assay’s control strategy. The external positive control includes one or more genomic regions targeted by your diagnostic assay. A failure of the external positive control indicates a failure of the reagents and/or reaction conditions associated with the amplification step of your assay. The laboratory needs to monitor the positive controls closely in order to prevent false negative results.
Every batch of specimens must include one or more negative control reactions. The negative control usually contains molecular grade water or reaction buffer. The negative control is set up during the nucleic acid extraction step and is handled and carried through the entire process just like all other specimens in the batch. The negative control is used to monitor the batch for cross-contamination and/or contamination of reagents. If the negative control generates a positive result, then the laboratory team must disqualify all results in that batch and trouble-shoot all steps to find the source(s) of contamination. Your laboratory must monitor the negative control(s) very closely in order to prevent false positive test results.
An assay control strategy like the one above will help your laboratory properly interpret patient results and ensure that the results are correct.
This post discussed some of the often-overlooked aspects of the analytic phase that are critical to high quality test results. In the next post, I will discuss some other ways to improve your laboratory’s performance in the analytic phase of testing. If your laboratory has questions or needs assistance with the analytic phase of diagnostic testing, reach out to an experienced CAP/CLIA Laboratory Director and CLIA laboratory builder (Contact).
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