AOAC Guidance on FA Immunoassay Validation (August 2023)

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claimed matrices. See Appendix N and Annex C of Appendix F for additional

descriptions of the POD model. (8, 12)

6.2.3.3.2.2. For each claimed matrix, the developer must prepare test materials with at least the following concentrations: blank, at the CDC, 0.5x CDC, and a high

level at least 1.5x CDC.

6.2.3.3.2.3. For test materials at 0.5x CDC and ≥ 1.5x CDC, perform 20 replicate tests of

each test material and calculate the POD at each concentration.

6.2.3.3.2.4. If the 0.5x CDC material produces a POD of 0%, an additional test material must be prepared at a concentration halfway between 0.5x CDC and the CDC (i.e., 0.75x CDC). The 0.75x CDC test material should then be analyzed with 20

replicate tests and the POD should be calculated.

6.2.3.3.2.5. If the 0.5x CDC material produces a POD of 100%, an additional test material at 0.25x CDC must be prepared, analyzed with 20 replicate tests, and the POD

calculated.

6.2.3.3.3. CDC Validation

6.2.3.3.3.1. In order to validate a CDC requiring a minimum POD of 0.9 with 95%

confidence, analyze a sufficient number of test portions for the bound of the 1-sided lower 95% confidence interval to be greater than 0.9 (as outlined in

Table A7 of Appendix F). (3)

6.2.3.3.3.2. For example, analyze 40 test portions with 40 positive results required, or

analyze 60 test portions, with 58 positive results required.

6.2.3.4. Data Analysis and Reporting

6.2.3.4.1. All results must be reported for all test portions analyzed.

6.2.3.4.2. Analyze the data for positive and negative responses, and prepare a table listing the matrix, concentration level, number of samples tested (N), number of positive samples, POD, 95% lower and upper confidence limits of the POD values. Use POD

data to prepare POD curve figures, as described in Appendix H. (14)

6.2.3.5. Acceptance Criteria

6.2.3.5.1. A POD of ≤ 0.1, with 95% confidence is required for blank test materials. 6.2.3.5.2. A POD of ≥ 0.9, with 95% confidence is required at the stated CDC.

6.2.4. Robustness Study

6.2.4.1. The Robustness study is intended to provide information on robustness and product

stability and consistency.

6.2.4.2. Test Materials

6.2.4.2.1. Spiked matrices are acceptable for lot-to-lot stability analysis and robustness analysis (except when varying extraction conditions). See Annex A for description of best

practices for spiked matrix preparation.

6.2.4.2.2. Incurred matrices may also be used. If sufficient quantities of incurred matrices have been prepared for the matrix study, these samples may also be used for the

robustness studies (i.e., separate incurred matrices are not required).