Pathology plays an integral role in the measurement and clinical validity phase of the evaluation pathway. Collaborating with relevant organisations, we work closely with the MIC to conduct rigorous validation and verification of proposed IVDs to ensure new IVD regulations are met. Within the Analytical and Clinical Validity Phase, the aim is to confirm that the proposed method has the capabilities and characteristics required for its purpose as stated in publication by manufacturers. This offers external verification of performance which is mutually beneficial for both the manufacturer and the NHS.
Assay suitability, from a technical perspective, is determined through a series of steps that make up the method validation process. Method validation tools are used to assess the claimed performance characteristics of the proposed assay and to determine accuracy, reliability, and reproducibility. We also explore cross reactivity with other analytes within the sample matrix and confirm analytical sensitivity. Available testing platforms within Pathology are used to conduct this process. Most test are completed within the Biochemistry discipline using biochemistry laboratory analysers.
When a new or existing method is in the scope for performance validation, a study plan is developed alongside a standard operating procedure (SOP) outlining the steps involved to analyse the new method.
Precision and Accuracy
- Precision studies involve investigating and establishing repeatability, intermediate precision and reproducibility of the proposed new method
- Is the assay precise, and how true to the target value does the results of each repeated run fall (accuracy)?
- Precision studies run a minimum of five replicates over five days as a starting point to assess variability both within and between each run
- Further replicates and repetitions are conducted if the data produced over the initial 5×5 is insufficient
- Robustness of method is investigated to establish if precision and accuracy are maintained with variable conditions such as different analyst, or different time of day
- From the data generated, mean, standard deviation and coefficient variation (CV) is calculated
Limit of Detection and Limit of Quantitation
- Limit of detection (LoD) is defined as the lowest concentration of an analyte that can be detected by the method
- The difference between ‘background’ and analyte signal is calculated by comparing the results from samples with known low concentrations and blank samples
- Limit of quantitation (LoQ) is the lowest concentration of the analyte that can be reliably detected
- This established by analysing samples with known concentrations of the analyte and establishing the minimum level at which the analyte can be quantified by acceptable accuracy and precision. This is interpreted through statistical analysis.
Specificity, Selectivity, Interference, Recovery
- To explore specificity and selectivity we aim to determine the extent at which the method can detect the target analyte within a sample matrix independent of the presence of interferences
- Interference and recovery experiments are performed to look at cross reactivity with other analytes
- Samples are spiked with known amounts of interference or analyte, and concentrations are measured and recovery calculated
- Manufacturers provide reportable ranges of assigned values that the method is able to detect to demonstrate linearity
- Test samples of unknown concentrations are run and plotted against the expected range values, and compared for precision and bias
- This comparison enables the biochemist to identify if the range declared by the manufacturer is met
Most of our testing is conducted on platforms that use immunoassay and spectrophotometry techniques for analysis. The Biochemistry Department at the Leeds Teaching Hospitals NHS Trust predominately uses Siemens equipment to perform routine and validation testing. Some of the platforms available include the automated Siemens Advia Centaur, which is a high throughput immunoassay analyser that uses acridium ester technology and chemiluminescent detection. The Advia XPT chemistry analysers use multiple assay types for analysis such as colorimetric and turbidametric assays, and end point assays with spectrophotometry techniques for detection. Further platforms are available to test methods that involve techniques such as nepholometry and RT PCR. All equipment used within the validation processed are calibrated and reagents and samples prepared as per assay manufacturer instruction.
The analytical procedures testing for precision, limit of quantitation and recovery are carried out by a HCPC registered Biomedical Scientist. These scientists are trained to perform laboratory techniques, run the analyser used for the IVD studies, conduct statistical analysis and interpret the resultant data. The results are then passed to Clinical Scientists who review the data and approve or reject the technical validity of the method.