AOAC Guidance on FA Immunoassay Validation (August 2023)

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6.2.4.2.3. If an environmental surface claim is included in the validation, the test materials

should be prepared as described in Annex B.

6.2.4.3. Robustness Study

6.2.4.3.1. The method developer, in conjunction with the AOAC project manager, is expected to make a good faith effort to determine which, and to what magnitude, parameters are most likely to vary in the hands of an end user. These parameters should be

tested in a screening study.

6.2.4.3.2. The robustness factors and levels should be used to generate a factorial or Plackett-

Burman study design. (10, 11)

6.2.4.3.3. Robustness should be evaluated with a blank test material and a test material at the

CDC.

6.2.4.3.4. Ten test portions of each test material should be analyzed for each factorial

combination.

6.2.4.3.5. The POD should be calculated for each factorial combination, and a generalized linear model (GLM) ANOVA analysis conducted on the POD values, for example, as

described by Crawley 2007. (15)

6.2.4.3.6. Data should be recorded in a table that describes each individual parameter variation, the individual results, the POD, and the POD 95% CI for each factorial combination.

The results of the regression should be reported in a separate table.

6.2.4.3.7. Any parameter change that has a significant effect on the POD (p<0.05 as determined

by ANOVA analysis) should be reported in the kit insert.

6.2.4.4. Product Consistency and Stability Study

6.2.4.4.1. If the test method is sold as a kit or device prepared in lots or batches, a product consistency and stability study must be performed to ensure that the performance of the product is consistent from lot-to-lot and over time under normal storage conditions for the shelf life of the product. Lot-to-lot consistency and product 6.2.4.4.2. A minimum of three separate product lots must be evaluated. The product lots should span the shelf life of the kit. For example, if the kit shelf life is 12 months, an approximately 12-month-old kit, six-month-old kit and recently produced kit should be evaluated. For an initial single lab validation, accelerated aging may be used if kits at the end of their shelf life are not available - if this is done, then lot-to-lot stability should still be performed across 3 recent lots. Kits should be aged using increased temperature storage as described in ASTM F1980-16 or CLSI EP25-A. Real time data is needed for validations such as AOAC Official Method applications, and stability can be measured in the same set of experiments.

prior to the first AOAC Performance Tested Method renewal.

6.2.4.4.3. Samples used in the evaluation should be made in any one matrix claimed for the method or using a stable control material. Samples should consist of a blank, as well

as a sample prepared at the CDC of the kit.

6.2.4.4.4. 20 tests of a single sample (one extract or one pooled extract) should be performed on each kit lot, following the method instructions. Data should be reported in a table listing the test kit lot, age of the lot (real time and accelerated), the individual