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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Evaluating Assay Precision

However, for a method developed in-house a higher level of proof is required to validate the method, in which case EPA2 would be the appropriate guideline to use. For this, longer-term assessment is required. The first replicate on day 1 is 2.

The user should ascertain that the imprecision of the candidate measurement procedure meets the criterion for allowable imprecision before beginning the evaluation.

T is best calculated in a spreadsheet and is given by:. This could be useful, for example, if the intent of the experiment was to estimate the bias of one laboratory in a system relative to another, or to the mean of the laboratories in a system. Table 3 shows the results of the same calculation for the remaining days.

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 be appropriate if the user is interested in estimating bias relative to that measurement procedure.

First, users rarely have access to the measurement procedure used by the manufacturer or authors of a publication as the comparative method for the published bias. Various materials may be used to complete the assessment with either protocol.


If an outlier is found the pair should be rejected and the cause investigated and resolved before repeating the run. 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 following example relates to the verification of performance of calcium according to EPA2 using a five day protocol. This is its fourth iteration, and although it retains much of its original approach, there were some significant changes in the A3 version.

Thus the variance of the daily means is:. If the mean concentration from the user’s experiment is within the verification interval, there is no statistically significant bias.

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.

Here’s a brief description of the protocol. 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.

For example, if the true standard deviations were actually exactly equal to their claimed counterparts, the calculated standard deviations would exceed their published counterparts fifty cpsi of the time in verification experiments.

If the estimated bias is less than allowable bias, the bias is acceptable. Thus we need to find the The width of the verification interval depends on the uncertainty of the target value of the reference material and the standard error of the calculated mean concentration from the experiment. The reader is referred to the CLSI documents for details. Similarly the within-laboratory precision is estimated by measuring a sample 20 times over cls days.


For example, on day 1 the average of the three values is 2.

Evaluating Assay Precision

The document includes tables to simplify the calculation of the verification limit. Sometimes the calculated standard deviations may exceed the published values, and yet the true standard deviations are less than the published values.

Estimation of Bias Because the precision experiment has so many csli measurements, collected over several days, results from the precision experiment may be used to make a reliable estimate of the bias of the cls procedure relative to the assigned target values of the sample materials used in the experiment. Australasian Association of Clinical Biochemists Website. Precision claims by a manufacturer should be tested at at-least two levels, by running three replicates over five days.

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. 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 quality control.

Dr Douglas Chesher e-mail: For the purposes of this discussion reproducibility will not be considered, as it involves multiple laboratories. When undertaking the assessment the data must be assessed for outliers, which are considered to be present if the absolute difference between replicates exceeds 5.