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HIV Drug Resistance Training. Module 7: HIV Genotyping Assay Validation . Topics. Getting Ready to Validate Validation Concepts Validation Procedures. Objectives. Describe principles behind validation procedures.

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hiv drug resistance training

HIV Drug Resistance Training

Module 7:

HIV Genotyping Assay Validation

  • Getting Ready to Validate
  • Validation Concepts
  • Validation Procedures
  • Describe principles behind validation procedures.
  • Identify the procedures needed to gain confidence that the results are accurate and reproducible.
  • Given specific laboratory, customize procedures.
getting ready to validate
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?

validation of genotyping assay performance
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.
minimum requirements
Minimum Requirements

Minimal requirements for an assay validation to establish/confirm analytical performance characteristics:






preparation procedures
Preparation: Procedures
  • Standard Operating Procedures (SOPs)
    • For what?
    • How?
  • Quality control (QC) SOPs
    • For what?
  • Personnel?
preparation validation protocol
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
when to repeat validation procedures
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
  • Why is it important to validate assays?
  • What should you do to prepare for validation?
  • When do you need to validate?
validation concepts
validation concepts

What criteria are we measuring against?

components of genotyping assay validation
Components of Genotyping Assay Validation
  • Accuracy
  • Precision/Reproducibility
  • Sensitivity for RT-PCR amplification
  • Sensitivity for detection of minority variants
  • Specificity
accuracy definition
Accuracy Definition
  • Are “known” mutations detected?
accuracy discussion
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
precision definition
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)
precision how close are all the results to each other
Precision: How close are all the results to each other?


(although inaccurate!)


(although sometimes accurate)

accuracy and precision
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.
accuracy and precision1

Precise: Yes

Accurate: No

Precise: Yes

Accurate: Yes

Accuracy and Precision

Precise: No

Accurate: No

  • 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
reproducibility and precision criteria
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
critical reagent lot to lot variability is a component of assay reproducibility
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)
sensitivity amplification
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
sensitivity detection of minority variants
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
dependence of sensitivity of detection of minor variants and input viral load
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
  • What criteria are we measuring against?
validation procedures
validation procedures

How can we set up validation procedures for our lab?

validation criteria example
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
specimen selection
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
validation procedure customization
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
  • How can we set up validation procedures for our lab?
  • What do we need to do to make sure we are correctly validating our assays?
  • Getting Ready to Validate
  • Validation Concepts
  • Validation Procedures