1. 1 HIV Drug Resistance Training Module 7: HIV Genotyping Assay Validation

2. 2 Topics Getting Ready to Validate Validation Concepts Validation Procedures

3. 3 Objectives Describe principles behind validation procedures. Identify the procedures needed to gain confidence that the results are accurate and reproducible. Given specific laboratory, customize procedures.

4. 4 getting ready to validate Why is it important to validate assays? What should you do to prepare for validation? When do you need to validate?

5. 5 Validation of Genotyping Assay Performance Helps ensure quality results Easier to compare results from labs using different methods To establish performance characteristics used to compare assays Required prior to testing any samples for WHO Validation is assay-specific –For instance, plasma vs. DBS sensitivity could be different, and extractions steps are not shared, so these methods should be validated separately.

6. 6 Minimum Requirements Minimal requirements for an assay validation to establish/confirm analytical performance characteristics: –Sensitivity –Specificity –Accuracy –Precision –Reproducibility

7. 7 Preparation: Procedures Standard Operating Procedures (SOPs) –For what? –How? Quality control (QC) SOPs –For what? Personnel?

8. 8 Preparation: Validation Protocol Written validation protocol finalized before results are analyzed –No changes to protocol allowed once validation experiments started Determine acceptance criteria for each section in advance Validation performed under the same conditions (facility, equipment, reagents, personnel) that will be used for real samples

9. 9 When to Repeat Validation Procedures If there is failure to pass any criteria: –Document corrective action and re-validate –Do NOT simply repeat experiments with same assay If procedure changes: –Minor changes equivalency testing –Major changes re-validation for in-house assays: Primer modifications and replacement Change RT and/or PCR enzymes –See module 11, QA/QC

10. 10 Discussion Why is it important to validate assays? What should you do to prepare for validation? When do you need to validate?

11. 11 validation concepts What criteria are we measuring against?

12. 12 Components of Genotyping Assay Validation Accuracy Precision/Reproducibility Sensitivity for RT-PCR amplification Sensitivity for detection of minority variants Specificity

13. 13 Accuracy Definition Are known mutations detected?

14. 14 AccurateInaccurate Accuracy: How close do we get to the expected (target) value or result.

15. 15 Accuracy Discussion Best tested using: –clonal virus or lab constructs (e.g. site-directed mutants) –very well-characterized, non-clonal sample (e.g. artificial 50/50 mixtures of clones) Test for: –Resistance-associated mutations –Likely to be frequently encountered Pro: Usually the easy to measure Con: Difficult to define criteria ahead of time for very new assays

16. 16 Precision Definition Ability to generate the same result on multiple aliquots of the same sample within a test run (intra-assay variability) –Fewer samples (e.g. 3 to 5), more replicates (e.g. 5 to 10)

17. 17 Precise (although inaccurate!) Imprecise (although sometimes accurate) Precision: How close are all the results to each other?

18. 18 Accuracy and Precision The degree of fluctuation in the measurements is indicative of the precision of the assay. The closeness of measurements to the true value is indicative of the accuracy of the assay. Quality Control is used to monitor both the precision and the accuracy of the assay in order to provide reliable results.

19. 19 Precise: No Accurate: No Accuracy and Precision Precise: Yes Accurate: No Precise: Yes Accurate: Yes

20. 20 Reproducibility Ability to generate the same result on multiple aliquots of the same sample in different test runs (inter-assay variability) –More samples (e.g. up to 40), fewer replicates (2 or 3) –Over time (e.g. 2 weeks) –Between operators –Among lots of critical reagents

21. 21 Reproducibility: When conditions change, how does that affect the results?

22. 22 Reproducibility and Precision Criteria Based on nucleotide sequence identity in: –Pairwise comparisons or –Comparison to a consensus sequence Mismatches may be considered a difference (if compatible e.g. A and R, or R and D, but not A and Y, or M and K) –Acceptance criteria may depend on complexity of samples tested Based on amino acid sequence identity –resistance-associated mutations can be analyzed separately

23. 23 Critical Reagent Lot-to-lot Variability is a Component of Assay Reproducibility Reagent vendors test products (e.g. PCR enzymes) using procedures different from HIVDR genotyping Cannot assume that performance is the same, between lots Incorporate at least 2 lots of critical reagents in validation experiments Perform lot-release testing as part of routine QC (see module 11)

24. 24 Sensitivity: Amplification Definition: What is the minimum viral load required to generate an accurate result? Considerations: –Importance placed on multiple subtypes varies with intended application –Overlaps with linearity if samples also genotyped and sequences compared –May or may not involve probing multiple variant detection (mixtures) at low VL

25. 25 Sensitivity: Detection of Minority Variants In samples with mixtures What % of the pool must minor variant be in order to be reliably detected? –Can separate out post-amplification steps (e.g. by mixing plasmids and sequencing vs. mixing viruses and amplifying then sequencing) –In reality, affected by viral load, though this is not often highlighted

26. 26 Dependence of Sensitivity of Detection of Minor Variants and Input Viral Load Assume that 200 µl plasma used for RNA extraction, 25% used for RT-PCR; RT successful for 20% of RNA molecules; minority variant present at 10% of total Viral load (copies/ml) RNA copies in RT rxn Amplifiable genomes Copy no. (minor variant) 100,0005,0001000100 10,00050010010 100050101

27. 27 Discussion What criteria are we measuring against?

28. 28 validation procedures How can we set up validation procedures for our lab?

29. 29 Validation Criteria (Example) Accuracy – 100% of known mutations must be detected Precision/Reproducibility – 90% of pairwise comparisons must be at least 98% identical, mixtures counted as difference Sensitivity for amplification – 95% of samples with viral loads between 500 and 1000 copies/ml must be amplified and successfully genotyped (n 10) Sensitivity for detection of minority variants –defined as the lowest % at which a mutation is detected in > 50% of replicate tests

30. 30 Specimen Selection Specimens used in the validation experiments should be chosen to test the specific aspect of assay performance (accuracy, reproducibility, etc.) Especially for precision and reproducibility, chose specimens that are similar to those that will be tested routinely (i.e. specimen type, genetic subtype, viral load range) Specimens should be well-characterized in pre- validation experiments before starting the actual validation

31. 31 Validation Procedure Customization Details of the variables to be considered are likely to be different in each lab –Physical set-up –Personnel (number of qualified analysts) –Degree of automation Validation protocols can be reviewed in consultation with other accredited labs or with WHO staff

32. 32 Discussion How can we set up validation procedures for our lab?

33. 33 Reflection What do we need to do to make sure we are correctly validating our assays?

34. 34 Summary Getting Ready to Validate Validation Concepts Validation Procedures