AOAC Guidance on FA Immunoassay Validation (August 2023)

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1.2.1.3.3. ANALYST/DAY Includes: Different operators, different times, different days, different teams, different environmental conditions in the lab, DIFFERENT CALIBRATION CURVES on different plates, different temperature. TP Includes: Test portion variation due to sampling, heterogeneity of the analytical sample (compositional and distributional), weighing variation, volume addition variation, extraction variation: time, temp, water bath fluctuation. This variance component will include everything that can happen within a set from weighing of the test portion until you are ready to take the ELISA includes: aliquot variation, heterogeneity in the extract, reagent pipetting variance, differences in coating of the wells, well-to-well sensitivity variation, rinsing issues, pipetting volumes, different optical density of each well, reader issues, timing of color development, how fast you pipet from start In order to make the software work, you need to give a name to each of the 4 variance components, with the understanding that there will be several sources of variation within each variance component category. I suppose you could call them Level 1, Level 2, Level 3, Level 4, but the usual way to do this is to take what you think is the most important source and use it as the “name” of the variance component – keeping in mind that the name is only a label and if you call the 3 rd level “Test Portion” that doesn’t mean that all those other sources are gone – this is just the Label we are using for convenience. (In these experiments, “Test Portion” will usually always include extraction sources as It’s critical to do the categorization of variance sources into variance components for 2 reasons: First, it is important to define terms, but more importantly, it will come in handy to determine if the factor is nested. Please note – if you only do the 3-factor experiments such as Design 1a or 1b, the variance components above labeled as “TP” and “ELISA” will be combined into 1 component. So, you may call that combined component “TP”, but it will contain all of the ELISA variance sources in addition to the other sources. (Maybe “TP” is not a good name for that in the 3-factor design.) Researchers are free to use any label for the name of the variance component, but this should always be understood that there are more sources of variation within a variance component than the one that is 1.3.1. Nested experiments are ones where you may have two or more factors involved and you have a hierarchical order of nesting of factors. This would be different from a factorial design where the factors are varied independently, and the conditions for one factor can be adjusted to be the same at all the other factor levels. In the case where we are doing a variance component analysis of a method take for example the factor “Test Portion.” Because each test portion is destroyed in the extraction, we can’t really have the exact same test portions for kit Lot 1 as kit Lot 2, so TP will always be a factor nested within some other higher level factor. In the same way, we pipet each extract into 2 wells on the 1.2.1.3.4. aliquot of the extract onto the ELISA plate. 1.2.1.3.5. to finish, different development times across the plate. 1.2.1.3.6. well.) 1.2.1.4. 1.2.1.5. used as the label. another factor?

1.3. What is a “Nested” experiment? When can we consider one factor to be “nested” within