Analytical sensitivity:
Analytical sensitivity is an assay’s ability to detect deficient concentrations of a given substance in a biological specimen. An assay that has 100% analytical sensitivity will not typically have any false negative results. Analytical sensitivity is often called the limit of detection (LoD). LoD is the actual concentration of an analyte in a specimen that can be consistently detected ≥ 95% of the time. The LoD may be represented as the number of genome copies, infectious dose, colony-forming units, plaque-forming units, etc., of the analyte that can be detected. An end-point dilution determines analytical sensitivity until the assay can no longer detect the target in more than 95% of the replicates. Analytical sensitivity can vary substantially for the same assay when different sample matrices are used. When setting up a dilution series, it is essential to use a diluent with qualities similar to the sample matrix (e.g., blood, CSF).
Analytical specificity:
Analytical specificity is an assay’s ability to detect the intended target. An assay with 100% analytical specificity will not typically have false positive results. Verifying that the assay’s primers are specific to the target is critical for PCR. Two components of analytical specificity are cross-reactivity and interference, which are discussed below.
- Cross-reactivity may occur when genetically related organisms are present in a patient specimen. These organisms mimic the intended target, which results in the assay’s primers cross-reacting or annealing to these genetically associated organisms. For example, a nasopharyngeal specimen collected for Bordetella pertussis may contain normal respiratory flora and other potential respiratory pathogens, such as Bordetella bronchiseptica. Suppose the specimen is to be tested for B. pertussis, and the assay primers are not specific to this target. In that case, the primers may anneal to other organisms, generating a false positive result.
- Interference may occur when a specimen is introduced to endogenous substances like hemoglobin, bilirubin, medications, etc., or exogenous inhibitory substances, such as hand cream, powdered gloves, serum separators, etc. These substances will inhibit the primers from binding to the intended target. If the target amplifies without the primers attaching, the target will not be detected, potentially generating a false negative result.
