One of the fundamental characteristics of any analytical method is the smallest concentration that can be reliably measured. A number of terms and concepts have been used to describe the lowest concentration an immunoassay can report, and this multiplicity of terms can be genuinely confusing. What follows is a discussion of some of these terms and how they relate to answering the fundamental question: What is the lowest concentration I can report with this assay?

Analytical sensitivity
The formal definition of analytical sensitivity is "the lowest concentration that can be distinguished from background noise." This concentration is properly termed the assay's detection limit, but it is most commonly referred to as sensitivity. Typically, this value is established by assaying replicates of a sample that is known to have no analyte present. Then the measured counts (CPS) from these replicates are used to calculate a mean and standard deviation (SD). The analytical sensitivity is determined as the concentration equivalent to the mean counts obtained from the zero sample plus 2 SD for immunometric assays, and the mean minus 2 SD for competitive assays. This is what is published in the "Analytical Sensitivity" section of IMMULITE® and IMMULITE® 2000 package inserts.

In the real world, analytical sensitivity has limited practical value. The real limitation is that, for any assay, imprecision increases very rapidly as concentration decreases. This phenomenon is readily apparent when looking at the assay's precision profile, which is a graphical representation of how the imprecision of an assay changes with the measured concentration. (See Figures 1 and 2 for examples.)



Figure 1. Representative DPC precision profile.





Figure 2. Representative DPC precision profile. Note that functional sensitivity in the 0.01-0.02 µIU/mL region, is required of a "third generation" TSH assay by definition.^1,2


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.

Having determined the day-to-day CV representing the clinically useful limit of reproducibility, the next step is to estimate the concentration at which the CV might reach this limit. On the basis of prior studies, package insert data, and estimates made from the assay's precision profile, Technical Services can usually help to identify a "target range" of concentrations bracketing the predetermined CV limit.

Ideally, this study should be performed using several undiluted patient samples, or pools of patient samples, with concentrations that span the target range. However, these samples may be difficult to obtain. Reasonable alternatives include patient samples diluted down to concentrations spanning the target range, or control materials in or near this range. If there is a need to dilute any type of sample for the study, the diluent used is critical. The routine sample diluents are intended only for diluting very high concentration samples; for some assays, they may have a measurable, though very low, apparent concentration. Use of these diluents can bias the results of the study.

The samples should be analyzed repeatedly over a number of different runs, ideally over a period of days or weeks, to assess the day-to-day precision. (A single run of 20 replicates does not provide a valid assessment of functional sensitivity.) Having collected the data, calculate the CV for each sample tested. The functional sensitivity is the concentration at which the CV reaches the predetermined limit. This concentration can be estimated from the study results by interpolation, if it doesn't happen to coincide with one of the levels tested.

Verifying the lower limit of the reportable range is one part of the process of verifying the entire reportable range. This is typically accomplished by performing replicate analysis on a series of three to five samples with known concentrations spanning the reportable range. These samples can be obtained using a single sample, with a concentration near the upper limit of the range, which is then diluted to give additional samples spanning the entire reportable range. The results obtained are evaluated for both reproducibility and recovery of expected values to determine that the assay's performance meets clinical usefulness needs across the reportable range.

Conclusion
So, why is the lower limit of the software reporting range 1.0 µg/dL (13 nmol/L) when the package insert says the sensitivity is 0.3 µg/dL (3.9 nmol/L)? In this example, the assay is a competitive assay and the imprecision of the assay exceeds clinically useful limits at a concentration well above the analytical sensitivity.

If the imprecision is such that you cannot say with certainty that results of, say, 0.4 µg/dL (5 nmol/L) and 0.7 µg/dL (9 nmol/L) are in fact different, might it not be better to report both as "< 1.0 µg/dL" ("< 13 nmol/L") rather than to risk having a physician interpret explicit results as showing a clinically meaningful difference?

Ultimately, it is usually not the assay's detection limit (analytical sensitivity) but rather the reproducibility of results which determines the lower limit of clinically reliable assay performance in routine practice.

References
1. Spencer CA. Thyroid profiling for the 1990s: free T4 estimate or sensitive TSH measurement. J Clin Immunoassay 1989; 12:82-9.

2. Spencer CA, et al. Interlaboratory/intermethod differences in functional sensitivity of immunometric assays of thyrotropin (TSH) and impact on reliability of measurement of subnormal concentrations of TSH. Clin Chem 1995;41:367-74. Reprint (catalog number ZD060) available from DPC on request.

*CLIA'88: the US Clinical Laboratory Improvement Act.