HIV Drug Resistance Training
Module 7:
HIV Genotyping Assay
Validation
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Topics
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Getting Ready to Validate
Validation Concepts
Validation Procedures
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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.
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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?
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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
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– For instance, plasma vs. DBS sensitivity could be
different, and extractions steps are not shared, so
these methods should be validated separately.
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Minimum Requirements

Minimal requirements for an assay validation to
establish/confirm analytical performance
characteristics:
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–
–
–
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Sensitivity
Specificity
Accuracy
Precision
Reproducibility
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Preparation: Procedures
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Standard Operating Procedures (SOPs)
– For what?
– How?
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Quality control (QC) SOPs
– For what?
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Personnel?
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Preparation: Validation Protocol
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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
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When to Repeat Validation Procedures
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If there is failure to pass any criteria:
– Document corrective action and re-validate
– Do NOT simply repeat experiments with same assay
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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
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Discussion
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Why is it important to validate assays?
What should you do to prepare for validation?
When do you need to validate?
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validation concepts
What criteria are we measuring against?
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Components of Genotyping Assay Validation
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Accuracy
Precision/Reproducibility
Sensitivity for RT-PCR amplification
Sensitivity for detection of minority variants
Specificity
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Accuracy Definition
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Are “known” mutations detected?
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Accuracy: How close do we get to the
expected (target) value or result.
Accurate
Inaccurate
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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
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Pro: Usually the easy to measure
Con: Difficult to define criteria ahead of time for
very new assays
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Precision Definition
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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)
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Precision: How close are all the results to
each other?
Precise
Imprecise
(although inaccurate!)
(although sometimes accurate)
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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.
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Accuracy and Precision
Precise: Yes
Accurate: No
Precise: Yes
Accurate: Yes
Precise: No
Accurate: No
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Reproducibility
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Ability to generate the same result on multiple
aliquots of the same sample in different test runs
(inter-assay variability)
–
–
–
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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
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Reproducibility: When conditions change,
how does that affect the results?
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Reproducibility and Precision Criteria
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Based on nucleotide sequence identity in:
– Pairwise comparisons or
– Comparison to a consensus sequence
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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
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Based on amino acid sequence identity
– “resistance-associated” mutations can be analyzed
separately
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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)
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Sensitivity: Amplification
Definition: What is the minimum viral load
required to generate an accurate result?
 Considerations:
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– 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
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Sensitivity: Detection of Minority Variants
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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
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Dependence of Sensitivity of Detection of Minor
Variants and Input Viral Load
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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)
100,000
5,000
1000
100
10,000
500
100
10
1000
50
10
1
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Discussion

What criteria are we measuring against?
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validation procedures
How can we set up validation procedures for our lab?
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Validation Criteria (Example)
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Accuracy
– 100% of known mutations must be detected
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Precision/Reproducibility
– ≥ 90% of pairwise comparisons must be at least 98%
identical, mixtures counted as difference
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Sensitivity for amplification
– ≥ 95% of samples with viral loads between 500 and
1000 copies/ml must be amplified and successfully
genotyped (n ≥ 10)
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Sensitivity for detection of minority variants
– defined as the lowest % at which a mutation is
detected in > 50% of replicate tests
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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 prevalidation experiments before starting the actual
validation
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Validation Procedure Customization
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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
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Validation protocols can be reviewed in
consultation with other accredited labs or with
WHO staff
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Discussion

How can we set up validation procedures for our
lab?
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Reflection

What do we need to do to make sure we are
correctly validating our assays?
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Summary
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Getting Ready to Validate
Validation Concepts
Validation Procedures
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