Not only at the analytical sensitivity, but even at concentrations significantly above it, imprecision may be so great that results do not reproduce well enough to be of real clinical utility. Consequently, analytical sensitivity does not typically represent the lowest measurable concentration that is clinically useful.
This limitation of analytical sensitivity has always been with us, for RIA and IMMULITE, and applies to all methods by all manufacturers. Since patient samples are not typically run in replicates, the lack of reproducibility may not be readily apparent in routine testing. However, the overall quality and usefulness of the results are affected. This is why the lower limit of the reporting range in the IMMULITE and IMMULITE 2000 software is often set at a concentration above the analytical sensitivity. DPC sets the reporting limits for every assay to what a comprehensive assessment suggests is the range of effective and reliable performance for the assay, relative to its intended clinical use.
The limitations of analytical sensitivity, for describing the lower limit of clinically effective assay performance, led to the development of another concept.
Functional sensitivity
About a decade ago, in reaction to the limited utility of analytical sensitivity as a measure of assay performance, a group of researchers evaluating TSH assays developed a concept they termed functional sensitivity.1 They defined it as "the lowest concentration at which an assay can report clinically useful results." Clinically useful results for TSH were deemed to be good accuracy with a day-to-day CV of not more than 20%. While this choice of CV limit was somewhat arbitrary, the researchers felt that, for TSH, a CV of 20% was the most imprecision that could be tolerated for clinical purposes.1
Since CV is the standard deviation expressed as a percentage of the mean, a CV of 20% implies the SD would be 20% of the mean. For a sample with a TSH concentration of 0.1 µIU/mL, for example, the range encompassing 95% of the expected results from repeat analysis would be ±40% (±2 SD), or 0.06 µIU/mL to 0.14 µIU/mL.
Although originally developed only for TSH assays, the concept of functional sensitivity and the use of a 20% CV as the limit of clinical usefulness have been widely applied to other immunoassays. The concept has gained acceptance because it provides the laboratory with an objective and clinically meaningful indication of the practical lower limit of an assay.
When developing a new assay, DPC uses essentially the same approach, evaluating both precision and accuracy to establish the concentrations at which the limits of clinical usefulness are likely to be reached. The software reporting range is based on this evaluation. For competitive assays especially, there is usually a significant difference between the analytical sensitivity and the lower reporting limit. The reporting range, as set in the IMMULITE and IMMULITE 2000 software, represents DPC's recommendation for the CLIA'88* "reportable range"-which is the concentration range over which assay performance is documented as valid.
Verifying assay performance
Currently, for laboratories using automated immunoassay systems in the US, the only sensitivity-related performance characteristic that CLIA'88 requires to be verified by the laboratory is the lower limit of the reportable range. Some laboratories may also choose to estimate the functional sensitivity of a new assay; and, historically, some have wanted to verify analytical sensitivity. Each of these assessments is a different experiment with distinct protocols and requirements. So the first step is to decide what is to be verified and then use the appropriate protocol and evaluate the data accordingly.
If a laboratory chooses to evaluate analytical sensitivity, the goal is typically to verify the value given for that performance measure in the package insert. It is essential that the sample used for an analytical sensitivity study be a true zero concentration sample with an appropriate sample matrix. Any other type of sample may bias the results. The usual protocol involves assaying 20 replicates of the zero sample, followed by calculating the mean and SD of the CPS. The analytical sensitivity is estimated as the concentration equal to the mean counts of the zero sample plus 2 SD for immunometric ("sandwich") assays like TSH, or minus 2 SD for competitive assays like T4. Technical Services can assist in calculating this concentration. This protocol yields an initial estimate, which is usually adequate for comparison with the analytical sensitivity listed in the package insert. However, multiple experiments encompassing several kit lots are necessary to obtain a robust and accurate assessment.
In assessing functional sensitivity, the aim is to determine the lowest concentration corresponding to a laboratory-specified goal for day-to-day (interassay) imprecision representing the limit of clinical usefulness for a given assay. Commonly, a CV of 20% has been used as the goal, based on the original application of the concept to TSH. However, this CV may not always be the most appropriate limit. For some assays, a CV greater than 20% may be consistent with clinically reliable and informative results, while for others, a CV less than 20% may represent the limit of clinical usefulness. The performance goal needs to be set for each assay, based on its intended clinical application.