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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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 cli for the within-run and between-run contributions to the overall imprecision of the measurement procedure.
A spreadsheet for assisting with the calculations described in this article is available from the AACB web-site. National Center for Biotechnology InformationU. 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. Part of the process of verifying or validating a method to confirm that it is suitable for use is an assessment of precision.
Verification of Precision EP15 first describes a precision verification experiment. In order to compare the estimated repeatability to a claimed value we can calculate the critical or verification value using the equation:. If an outlier is found the pair should be rejected and the cause investigated and resolved before repeating the run.
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 user must evaluate the estimated bias versus allowable bias.
Evaluating Assay Precision
This article has been cited by other articles in PMC. If the mean concentration from the user’s experiment is beyond the verification el15, statistically significant bias exists. 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.
Evaluation of precision performance of quantitative measurement methods. If the mean concentration from the user’s experiment is within the verification interval, there is no statistically significant bias. However, if the values achieved are greater than those reported by the manufacturer, a statistical test needs to be performed to determine whether this difference is statistically significant. EP15 first describes a precision verification experiment.
Note, some authors refer to total variation as just the between-run component instead of combined between-run and within-run shown above. 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.
Summary When evaluating the epp15 of a method it is necessary to assess the repeatability within-run and the total or within-laboratory precision. The only requirement is that the assigned value must be available.
CLSI/NCCLS: EPA2. User verification of performance for precision and trueness – ScienceOpen
Summing the square of the differences gives a total of 0. User verification of performance for precision and trueness; approved guideline.
If the user is evaluating a procedure for which there clsu 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.
Eep15 essentials to accomplish this were present in EP15 through all of its previous versions, but they are refined and combined in EPA3 to make a single experiment. 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.
CLSI document EPA2 describes the protocols that should be undertaken by the user to verify precision claims by a manufacturer. There were two problems with this approach.
For this, longer-term assessment is required.
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. If this is true then using the principle of analysis of variance components:. The assessment is performed on at least two levels, as precision can differ over the analytical range of an assay.
Table 2 shows the results of each of these calculations. 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. Cclsi 3 shows the results of the same calculation for the remaining days. The first step is to calculate the mean of the replicates for each day, then for each result subtract the mean for that day and ep1 the resultant value.
As alluded to above, EPA2 is generally used to verify that a method is performing as is claimed by the manufacturer.
Repeatability Verification Value In order to compare the estimated repeatability to a claimed value we can calculate the critical or verification value using the equation: Using the values from our example the mean of all the results is 1. Estimating Precision When evaluating the precision of an assay, the trivial approach for estimating repeatability for any given level is to perform 20 replicate analyses in a single run on a single day.
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. T is best calculated in a spreadsheet and is given by:. Estimation of Bias Because the precision experiment has so many replicate measurements, collected over several days, results from the precision experiment may be used to make a reliable estimate of the bias of the measurement procedure relative to the assigned target values of the sample materials used in the experiment.
EPA2 should be used to validate a method against user requirements, and is generally used by reagent and instrument suppliers to demonstrate the precision of their methods. Various materials may be used to complete the assessment with either protocol. The next step is to calculate the variance for the daily means s b 2 using the equation.
To allow for this possibility, the user calculates a “verification limit” based on the published standard deviation and the size of the user’s experiment. 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.
Evaluating Assay Precision
The repeatability and within-laboratory standard deviations are then compared to vlsi claimed or published standard deviations. Selection and analytical evaluation of methods with statistical techniques. For the purposes of this discussion reproducibility will not be considered, as it involves multiple laboratories. If the repeatability and within-laboratory Ep51 are less than that indicated by the manufacturer, then the user has demonstrated precision consistent with the claim and no further calculations are required.
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.