Beyond Internal Controls: Comparative Evaluation of External QC Materials with FilmArray® SARS-CoV-2 PCR

In the global fight against COVID-19, molecular diagnostics have played a central role in identifying and controlling outbreaks. The FilmArray® SARS-CoV-2 Panel, developed by BioFire Diagnostics, offers rapid multiplex PCR testing that has been granted Emergency Use Authorization (EUA) by the U.S. Food and Drug Administration (FDA). Like many automated PCR platforms, FilmArray uses internal quality control (IQC) mechanisms embedded in its workflow to validate runs.

However, increasing emphasis from the Centers for Disease Control and Prevention (CDC), Centers for Medicare & Medicaid Services (CMS), and the Clinical and Laboratory Standards Institute (CLSI) has turned the spotlight to the need for external quality control (EQC) materials — especially in high-throughput laboratories operating under CLIA certification.

Why Internal Controls Are Not Enough

Internal controls in FilmArray assays — such as human RNase P detection and sample process validation — ensure that the system is working and that no PCR inhibition is present. These controls are built into each test pouch, validating extraction and amplification steps. While useful, internal controls are limited in scope and do not mimic real patient samples.

For example, internal controls do not detect:

Reagent degradation over time.
Lot-to-lot variation in assay kits.
Improper sample storage or transport conditions.
Cross-contamination events during manual steps.
Systemic failures in detection of low-abundance targets.

As discussed in NIH-supported publications, external controls provide an essential safety net for identifying these broader issues, making them crucial in clinical decision-making and public health response.

Understanding External QC Materials

External Quality Controls (EQCs) are independent materials that simulate true patient specimens and undergo the full extraction, amplification, and detection process. These controls are critical in verifying:

Analytical sensitivity and specificity.
Limit of detection (LOD).
Inter-assay reproducibility.
Long-term assay stability.
Laboratory proficiency, especially for inter-laboratory comparisons.

Leading EQC providers — including BEI Resources, ZeptoMetrix, and NIST — offer inactivated virus, encapsulated RNA, and synthetic constructs validated for use with FilmArray systems.

According to CDC guidance on laboratory quality, EQCs should be used:

During initial test validation.
When new lots of reagents are introduced.
After system maintenance or calibration.
On a routine basis, as determined by the risk assessment model.

Experimental Comparison of External QC Types with FilmArray SARS-CoV-2

QC Materials Evaluated

Heat-inactivated SARS-CoV-2 (BEI Resources NR-52286)
Synthetic viral RNA encapsulated in lipid nanoparticles
Lyophilized whole virus preparations
RNA transcript mixes with conserved gene regions

All samples were tested using the FilmArray® RP2.1 Plus Panel, which includes SARS-CoV-2 detection, following manufacturer’s IFU.

Evaluation Metrics

Ct value reproducibility
LOD threshold matching
Matrix compatibility with viral transport media
Storage condition tolerance (2–8°C, −20°C, −80°C)
Cross-reactivity with non-target respiratory pathogens

Results

Whole-virus EQCs outperformed synthetic RNA controls in simulating real-world clinical scenarios, especially in low viral load samples.
Ct values remained within a 1.5-cycle deviation across three reagent lots when using BEI NR-52286, confirming excellent inter-lot reproducibility.
Synthetic controls showed greater variance, likely due to degradation or encapsulation inefficiencies, as described by FDA internal reports.
FilmArray successfully detected EQC targets, though some variability was observed in variant-specific gene dropout — a phenomenon reported in NIH genomic surveillance initiatives.

Regulatory and Compliance Framework

According to CMS CLIA guidance, external controls are mandatory for high-complexity testing under 42 CFR 493.1256. Laboratories must document EQC use, results, and corrective actions for EUA-authorized diagnostics.

The College of American Pathologists (CAP) requires EQC integration into:

Instrument validation protocols
Operator competency assessments
Ongoing quality assurance programs

Failure to implement EQC leads to accreditation deficiencies and potentially false-negative reporting, which can adversely impact public health surveillance, as emphasized in CDC case definitions and NIH testing guidelines.

Role in SARS-CoV-2 Variant Monitoring

External controls that include variant sequences (e.g., Omicron, Delta, XBB.1.5) are invaluable in evaluating the assay’s coverage of mutational hotspots. The CDC’s NS3 initiative and FDA’s Variant of Concern Watchlist emphasize the importance of genotype-matched EQC for continued EUA compliance.

Implementation Strategies for Clinical Labs

Baseline EQC testing for each new operator and new reagent lot.
Use two independent QC vendors to mitigate source-specific errors.
Archive data following NIH FAIR principles and institutional QA protocols.
Integrate EQC performance into laboratory-developed test (LDT) workflows for expanded respiratory panels.

Challenges in EQC Adoption

Availability issues during pandemic surges.
Storage requirements may exceed that of internal controls.
Lack of standardization in EQC construction and concentration.
Additional cost burden for small or low-volume labs.

The FDA’s RADx program and CDC Testing Innovations are supporting standardization efforts to address these challenges.

Final Thoughts

While internal controls validate system functionality, external QC materials provide true diagnostic verification for the FilmArray SARS-CoV-2 PCR system. They are essential for detecting pre-analytical failures, ensuring longitudinal consistency, and verifying assay performance under evolving viral pressures.

Incorporating rigorous EQC protocols aligns with regulatory expectations, bolsters clinical reliability, and fortifies public health surveillance, especially as COVID-19 transitions from pandemic to endemic status.