Neill Carey, Ph. This is its fourth iteration, and although it retains much of its original approach, there were some significant changes in the A3 version. The 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. The user should ascertain that the imprecision of the candidate measurement procedure meets the criterion for allowable imprecision before beginning the evaluation. Verification of Precision EP15 first describes a precision verification 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.
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Requests to do so should be addressed to the Editor. Summary When evaluating the precision of a method it is necessary to assess the repeatability within-run and the total or within-laboratory precision. It is insufficient to assess repeatability in a single run. CLSI document EPA2 describes the protocols that should be undertaken by the user to verify precision claims by a manufacturer. Precision claims by a manufacturer should be tested at at-least two levels, by running three replicates over five days.
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 the purposes of this discussion reproducibility will not be considered, as it involves multiple laboratories.
Instead total precision within a laboratory within-laboratory precision will be assessed. While the term precision relates to the concept of variation around a central value, imprecision is actually what is measured. For a normal distribution the measure of imprecision is the standard deviation SD.
Alternatively one can use the variance, which is simply the square of the SD. 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. Note, some authors refer to total variation as just the between-run component instead of combined between-run and within-run shown above.
Care must be taken in knowing which term is being referred to. 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.
For n measurements we have: 1 The coefficient of variation CV is defined as: 3 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. Similarly the within-laboratory precision is estimated by measuring a sample 20 times over multiple days.
Unfortunately this approach is insufficient, as it tends to under-estimate repeatability, as the operating conditions in effect at the time may not reflect usual operating parameters. 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. 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.
Various materials may be used to complete the assessment with either protocol. These include pooled patient samples, quality control material, or commercial standard material with known values. When using quality control samples, these should be different to those used to ensure the instrument is in control at the time of the assessment.
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. For this, longer-term assessment is required. Assessment Protocols The EPA2 protocol recommends that: The assessment is performed on at least two levels, as precision can differ over the analytical range of an assay. Each level is run in duplicate, with two runs per day over 20 days, and each run separated by a minimum of two hours.
There should be at least one quality control QC sample in each run. If QC material is being used for the precision assessment, it should be different to that used to control the assay. The order of analysis of test materials and QC for each run or day should be changed. To simulate actual operation, include at least ten patient samples in each run.
The EPA2 protocol is similar except that the experiment is undertaken with three replicates over five days for at least two levels. The reader is referred to the CLSI documents for details. If an outlier is found the pair should be rejected and the cause investigated and resolved before repeating the run. The figure of 5. Estimation of Repeatability and Within-Laboratory Precision The following example relates to the verification of performance of calcium according to EPA2 using a five day protocol.
For the purposes of this example the results of only a single level are shown Table 1.
Global Laboratory Standards for a Healthier World
Evaluating Assay Precision