Method Validation and
Verification: An Overview
Patricia Hanson, Biological Administrator I
Florida Department of Agriculture and
Consumer Services, Food Safety
Microbiology Laboratory
Introduction
• 17 years in the microbiology section of the
Florida Department of Agriculture and
Consumer Services, Food Laboratory
• Laboratory moved from primarily cultural
methods in 1998 to a leader in the
implementation of new technology in 2015
• Numerous partnerships with state, federal and
industrial customers to validate and
implement new methods
Goals
• Distinguish between method verification and
method validation
• Based on our laboratory’s experience, identify
the critical components of both a method
verification and a method validation
• Offer insight to approaches we have found
useful, specifically in the microbiology section
• Share lessons learned
Verification vs Validation
• Verification is a demonstration that your
laboratory can perform a Standard Method or
other well documented method and produce
acceptable results
• Validation is both a demonstration that your
lab can run the method and that the method
or change to a method is fit for purpose
Aspects of a Verification
• Run entire method, start to finish, exactly as it
is written
• Include both positive and negative samples
• Represent all matrix types
• Be spiked at a level that is fit for the purpose
of the method if spiked samples are used
Verifications can be ongoing….
• For the purpose of this discussion,
“Verification” is being used to describe what
the laboratory does prior to implementing a
method in the laboratory
• The term “Verification” is often also used to
describe the ongoing process of verifying a
method with each run – in our lab this it the
use of Process Controls, Cultural Controls, and
Media Controls
Validation
• Laboratory developed method
• Matrix Extensions
• ANY technically significant change to a
Standard Method or previously validated
method
Overview of an Existing Guideline
• Level One: Urgent Use, Single Lab Validation
• Level Two: Single Lab Validation
• Level Three: Multi-Lab Validation
• Level Four: Full Collaborative Study
Level 2: Single Lab Validation
Highlights
• 50 different strains of target organism, if available
• 30 different strains of “nearest neighbor” organisms
• At least two target analyte levels
• 20 replicates per food type
per analyte level
• Perform side by side if possible
Aspect of a Verification
• Be extensive enough to show that the method
is fit for the intended use and meets the
customer’s needs
What is “Extensive Enough”
Items to be considered:
 Matrix: number and types
 Target: source and levels
 Method Evaluation: side by side or use of
standard reference material
When considering these items during the
development of the validation, keep in mind what
is needed to show the method is fit for the
intended use and meets the customer needs.
Matrices
• Validation or verification needs to be
performed on each food type
• Meat (raw, smoked, cooked, etc)
• Vegetable (whole, cut, sprout, etc)
• Dairy (liquid, solid, etc)
• Other (spices, candies, etc)
Replicates
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•
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Exact number depends on method
Verification, normally triplicate
Validation, normally six replicates per level
Important to do the same number of
replicates of “un-spiked”
Naturally Contaminated Samples
• Naturally contaminated pros and cons
• Best test of the method
• Level of analyte is unknown
• Little or no control of analyte level
Spiked Samples
• Spiked sample pros and cons
• Ability to control organism level
• Methods often perform differently on spiked
samples than on naturally contaminated
samples
Aging of Spiked Cultures
• Spiking samples 48-72 hours prior to initiation
of testing can better represent naturally
occurring contamination
• Sometimes the cells are so stressed, they die
off
• Consider the method or part of method being
validated and type of validation
Selection of Organisms
• Select at least one organism for each target
• “Nearest Neighbor” organisms – not often
used but good to consider
“Side by Side” Validations
• Normally involves running the method being
validated side by side with a Standard or established
method
• Twice the work but very valuable for determining if
the “new” method is equivalent or better than the
Standard or established method
• Best choice when changing part of a method – for
example a new enrichment broth but same screening
method
How Much Confirmation?
• Screening Method Validations: Confirm
everything
• You can get by with an abbreviated
confirmation, for example just biochemical
panel and/or serology but do enough to show
you “got out what you put in.”
Why Do So Many Confirmations?
• Ensure you can confirm samples that screen
positive
• Ensure samples you can confirm screen positive
• Spiked samples that don’t screen positive and
don’t confirm point to limitations of the method
or problems with the spiking procedure
Data Evaluation
• The measures used to evaluate data depend on
the purpose and type of the method and the
needs of the customer
Qualitative Data Evaluation
• Detection Limits – can the method detect the target
at the levels required by the customer?
• Specificity – can the method detect the target in the
matrix?
• Selectivity – can the method differentiate between
the target and other analytes in the matrix?
• Robustness – can the method be repeated on the
same sample with the same results?
Quantitative Data Evaluation
• Detection Limits, Specificity, Selectivity, and
Robustness
• Precision – Does the method produce the
same results as established method?
• Linearity – Does the method produce
proportional results with in the customer’s
target range?
Personnel Considerations
• Personnel designing and approving verifications
and validations should be knowledgeable about
the technology as well as the needs of the lab
Personnel Considerations
• Personnel carrying out verification and
validations should be competent in all
techniques
• Work on verification and validation studies
can be used to demonstrate competency but
should not be used as training
Pit-falls of Method Validation
• Repeating tests until you get the result you
want
• Drifting from method validation into method
development
• Adjusting criteria
for acceptance AFTER
the data is obtained
Overcoming Pit-falls
• Have a well thought out, documented and
approved plan and stick to it
• Go back and start over, incorporating any
changes into new validation
• All data should still be included in evaluation
Documentation
•
•
•
•
•
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Approval of plan prior to running
Raw Data
Interpretation of raw data
Final approval from customer
Reference validation in SOP
If SOP is a modified Standard Method, list
deviations from the Standard Method in a
section in the SOP
References
• U. S. Department of Health and Human Services, Food
and Drug Administration (2014, August 29). Volume II –
Methods, Method Verification and Validation ORALAB.5.4.5. Retrieved from
http://www.fda.gov/ScienceResearch/FieldScience/ucm1
71877.htm
• U. S. Department of Health and Human Services, Food
and Drug Administration (2011, September 8).
Guidelines for the Validation of Analytical Methods for
the Detection of Microbial Pathogens in Foods. Retrieved
from
http://www.fda.gov/downloads/ScienceResearch/FieldSc
ience/UCM273418.pdf
Questions