The EPA2 protocol from CLSI. • Uses control material with assigned concentration (e g from external quality control) or certified reference materials. We are pleased to have a guest essay explaining the latest in Method Verification , specifically the newest version of the CLSI guideline EP15 on Method. CLSI document EPA2 describes the protocols that should be undertaken by the user to verify precision claims by a manufacturer. Precision claims by a.

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Using the values from our example the mean of all the results is 1. The experiment produces at least 25 replicates collected over at least 5 days for each sample material. CLSI now uses the term within-laboratory precision to denote the total precision within the same facility using the same equipment 1 and this term will be used for this concept throughout this paper.

If this is true then using the principle of analysis of variance components:. National Center for Biotechnology InformationU. The repeatability previously termed “within-run” and the within-laboratory previously termed “total” standard deviations are calculated by an analysis of variance technique ANOVA that properly accounts for the within-run and between-run contributions to the overall imprecision of the measurement procedure.

Typically, there is no way to estimate the uncertainty of the “assayed” values, which is needed to determine if the calculated bias is statistically significant. Verification of Precision EP15 first describes a precision verification experiment. Internationally recognized high order reference materials, such as a material from the U. The user should ascertain that the imprecision of the candidate measurement procedure meets the criterion for allowable imprecision before beginning the evaluation.

For a normal distribution the measure of imprecision is the standard deviation SD.

Evaluating Assay Precision

Instead total precision within a laboratory within-laboratory precision will be assessed. Patient samples or control materials which have been repeatedly assayed with a measurement procedure felt to be substantially equivalent to the measurement procedure being evaluated may x2 appropriate if the user is interested in estimating bias relative to that measurement procedure.


Patient samples, reference materials, proficiency testing samples, or control materials may be used as the test samples, provided there is sufficient sample material for testing each sample five times per run for five to seven runs. The document includes tables to simplify the calculation of the verification limit.

Guest Essay

Reproducibility is at the other extreme and refers to the closeness of agreement between results of successive measurements obtained under changed conditions time, operators, calibrators, reagents, and laboratory. For this, longer-term assessment is required. Note, some authors refer to total variation as just the between-run component instead of combined between-run and within-run shown above. As the period of assessment is quite short, the total SD or within-laboratory SD derived from these experiments should not generally be used to define acceptability limits for internal sp15 control.

If the repeatability and within-laboratory SD are less than that indicated by the manufacturer, then the user has demonstrated precision consistent with the claim and no further calculations are required. The most significant change is the creation of a relatively simple experiment that gives reliable estimates of a measurement procedure’s imprecision and its bias.

Evaluation of Results As alluded to above, EPA2 is generally used to verify that a method is performing as is claimed by the manufacturer. The next step is to calculate the variance for the daily means s b 2 using the equation. Elsevier Saunders; St Louis: Linnet K, Boyd JC. Previous versions of EP15 included a small comparison experiment, involving 20 patient samples, which was to be used to verify a manufacturer’s claimed bias.

Table 2 shows the results of each of these calculations.

Evaluating Assay Precision

Dr Douglas Chesher e-mail: Using the example data and assuming the claimed repeatability is an improbable CV of 1. Acknowledge Committee Members The EPA3 document development committee was team of experts who worked together well. For the purposes of this discussion reproducibility will not be considered, as it involves multiple laboratories. Author information Copyright and License information Disclaimer.


The EPA3 document development committee was team of experts who worked together well. Summing the square of the differences gives a total of 0. If the calculated standard deviation is less than the verification limit, it is not statistically significantly larger than the published standard deviation, and the user has verified the published precision.

The user needs access to software to do the ANOVA calculations, but they are available in Excel, Minitab, Analyze-it, and other software packages that do statistical calculations. For bias relative to the quality control peer group, quality control materials with peer group values for the measurement procedure are appropriate.

T is best calculated in a spreadsheet and is given by:. Here’s a brief description of the protocol. The figure of 5. It is generally assumed in the laboratory that the variation associated with repeated analysis will follow a normal distribution, also known as the Laplace-Gaussian or Gaussian distribution.

Tools, Technologies and Training for Healthcare Laboratories. Various materials may be used to complete the assessment with either protocol. If the user is evaluating a procedure for which there are manufacturer’s precision claims, or published precision results, that were developed using CLSI EP5, the user can verify the published precision in an experiment lasting as few as five days.

If the calculated precision exceeds the verification limit, the calculated standard deviation is statistically significantly larger than the published standard deviation, and the user has failed to verify the published imprecision.

For the purposes of this example the results of only a single level are shown Table 1. Sometimes the calculated standard deviations may exceed the published values, and yet the true standard deviations are less than the published values.