The BioFire® FilmArray® Respiratory Pathogen PCR Panel is a highly efficient diagnostic tool designed for the detection of respiratory pathogens. This multiplex PCR system enables the rapid identification of a wide array of respiratory pathogens from a single sample. To ensure the accuracy and reliability of results, both internal and external controls are critical components of the assay. These controls play a vital role in confirming the performance of the assay, identifying potential issues, and ensuring high-quality results. This article discusses the role of these controls in the BioFire® FilmArray® Respiratory Pathogen PCR Panel, with an emphasis on best practices for quality assurance.
The Importance of Controls in PCR Diagnostics
In molecular diagnostics, including PCR-based systems like the BioFire® FilmArray® Respiratory Pathogen PCR Panel, controls are essential for confirming the integrity of the assay. Controls are used to monitor the performance of the PCR process, ensuring that all steps—such as amplification, detection, and data analysis—proceed correctly. In this context, controls are divided into two broad categories: internal controls and external controls.
Internal controls are included in each reaction to monitor the integrity of the sample and PCR process, while external controls are run separately to verify the system’s overall performance. Both types of controls are integral to ensuring that the PCR test delivers reliable, accurate, and reproducible results. According to the CDC’s guidelines on molecular testing, controls help to detect potential problems such as contamination, improper reagent use, or equipment failure.
Internal Controls in the BioFire® FilmArray® Respiratory Pathogen PCR Panel
Internal controls are included directly in the PCR reaction. These controls are typically synthetic nucleic acids or other substances that mimic the target pathogen. The inclusion of these controls serves multiple purposes in quality assurance:
-
Sample Integrity Verification: Internal controls ensure that the sample has been properly collected, stored, and transported, which is essential for confirming that the results are not compromised by sample degradation. For example, the inclusion of a process control, such as an RNA control, ensures that the sample’s RNA has not been degraded before testing. For more details on RNA quality controls, see this NIH article on RNA integrity.
-
Amplification Efficiency Monitoring: Internal controls monitor the efficiency of the PCR amplification process, ensuring that the reagents and equipment are working correctly. The control’s presence in the reaction helps verify that all PCR reagents are functioning, including primers and polymerases. The FDA’s guidance on PCR assay validation outlines the need for internal controls to verify amplification efficiency.
-
Inhibition Detection: Inhibition of the PCR reaction can lead to false-negative results, making the inclusion of an internal control essential for detecting any inhibitory substances present in the sample. NCBI’s article on PCR inhibitors provides more information on common PCR inhibitors and how internal controls help detect them.
-
Cross-contamination Prevention: Internal controls also help identify contamination that might occur during the sample preparation or PCR amplification steps. If the control sequence amplifies when it should not, it is an indication that cross-contamination may have occurred. The CDC’s resource on contamination prevention provides valuable insights into minimizing cross-contamination in PCR testing.
External Controls in the BioFire® FilmArray® Respiratory Pathogen PCR Panel
External controls, while not included in the reaction itself, are critical for verifying the overall performance of the BioFire® FilmArray® system. These controls typically involve the use of known positive and negative samples, which are tested alongside patient samples to validate the performance of the entire system.
-
Verification of Assay Performance: External controls are used to confirm that the PCR system, including the instrument and software, is functioning correctly. Positive controls contain known respiratory pathogens, while negative controls are free from pathogens. According to CDC’s recommendations for diagnostic testing, external controls verify that the system is capable of detecting the pathogens as expected.
-
Validation of Reagents and Equipment: External controls are particularly useful in detecting problems with the reagents or equipment. For instance, if a positive control fails to amplify or a negative control shows amplification, this could indicate a problem with the reagents or instrumentation. The FDA guidelines recommend using external controls to monitor system performance.
-
Confirmation of System Accuracy: External controls allow for the confirmation of test accuracy by testing samples with known pathogen content. This ensures that the PCR system’s readings align with known outcomes. Regular testing of external controls is essential for maintaining confidence in the system’s ability to detect pathogens accurately.
-
Training and Calibration: The use of external controls is also important for training laboratory personnel and ensuring proper calibration of the system. By regularly running external controls, laboratories can detect any drift in system performance over time. More on calibration procedures can be found in this resource from NIH.
Best Practices for Control Use in PCR Testing
To ensure the proper use of internal and external controls in the BioFire® FilmArray® Respiratory Pathogen PCR Panel, laboratories should adhere to the following best practices:
-
Routine Validation: Regular use of both internal and external controls is essential for routine system validation. These controls should be included with each batch of patient samples to verify both sample integrity and system performance.
-
Documentation: Laboratories should maintain detailed records of control performance, including any failures or anomalies. This documentation is critical for troubleshooting and for regulatory compliance. According to CDC’s laboratory quality assurance guidelines, proper documentation ensures that any performance issues are detected and resolved promptly.
-
Control Frequency: While internal controls should be included with every sample, external controls should be run periodically to validate the overall system. The frequency of external control testing may depend on the laboratory’s internal policies or the manufacturer’s recommendations.
-
Troubleshooting: In the event of control failure, it is important to have troubleshooting procedures in place. Possible issues include reagent degradation, equipment malfunction, or incorrect sample preparation. The FDA’s troubleshooting guide provides guidance on addressing common PCR testing issues.
-
Training: Laboratory staff should be well-trained in the proper handling of controls, including how to interpret control results and respond to failures. Training ensures that the testing process remains consistent and that quality assurance standards are met.
Conclusion
Internal and external controls are integral to ensuring the accuracy, reliability, and performance of the BioFire® FilmArray® Respiratory Pathogen PCR Panel. These controls help to verify sample integrity, assay efficiency, and system performance, providing confidence in the diagnostic results. By adhering to best practices for control use, laboratories can maintain high-quality testing standards and ensure the reliability of their results. For more details on quality assurance and PCR testing, refer to FDA guidelines on diagnostic testing and CDC recommendations on PCR testing.