Considerations for Clinical Application Selection and Performance Validation of Liquid Biopsy Reagents

Clarify reagent applicability in conjunction with the detection platform.

Before selecting liquid biopsy reagents, clinical laboratories should first determine whether the intended detection platform is real-time fluorescence quantitative PCR, digital PCR, or high-throughput sequencing. Different platforms have different requirements for nucleic acid input amount, library concentration, fragment distribution, and adapter structure. In general, it is recommended to prioritize kits that are compatible with existing equipment, have clear operating procedures, and contain complete components, and to verify the sample types, storage conditions, and compatible instrument models in the instructions.

If the laboratory has not yet established an NGS platform, it may first use digital PCR or fluorescence PCR-based liquid biopsy reagents to perform detection of known targets, and then gradually expand to multi-gene panels. When switching platforms, the nucleic acid extraction efficiency, library preparation yield, and sequencing data quality should be reassessed to avoid simply applying the original parameters.

Points to note in sample collection and preprocessing.

The stability of liquid biopsy samples is greatly affected by time, temperature, and degree of hemolysis. Blood collection tubes generally need to contain cell-free DNA preservatives or cell-stabilizing components. After blood collection, centrifugation should be completed within the specified time. If plasma is to be stored for the long term after separation, it is recommended to aliquot it and place it at -70°C or below. For samples such as urine and pleural or ascitic fluid, attention should be paid to cell sediment, microbial contamination, and nucleic acid degradation.

In the nucleic acid extraction step, laboratories may compare the recovery rate, purity, and processing time of different extraction reagents. For low-frequency mutation detection, the integrity and purity of the extracted product have a relatively large impact on subsequent results. Generally, the A260/A280 ratio is required to be within a reasonable range, with no residual alcohol or salt ions. It is recommended to set up parallel comparisons when changing batches.

Analytical performance verification and internal quality control.

Before liquid biopsy reagents are put into clinical testing, laboratories should complete the necessary performance verification based on their own conditions. Common items include precision, accuracy, limit of detection, analytical specificity, and anti-interference capability. Verification samples may use reference materials with known mutation frequencies or external quality assessment samples, but overall performance should not be inferred from the results of individual samples.

In routine operation, it is recommended to set up positive quality controls, weak positive quality controls, and negative controls to monitor inter-batch differences in reagents and instrument status. For NGS reagents, attention should also be paid to sequencing depth, uniformity of target region coverage, base quality values, and reproducibility of mutation abundance. If quality control deviations occur, reporting should be suspended and the cause investigated.

When liquid biopsy results are used for clinical decision-making, they should be comprehensively judged in combination with imaging, histopathology, and other laboratory indicators, and should not be used alone as a basis for diagnosis. The intended use and limitations in the reagent instructions should also be truthfully communicated to relevant users.