Method validation and method verification

Method validation and method verification

The ISO 16140 series is dedicated to the validation and verification of microbiological methodsThese International Standards are designed to help food and feed testing laboratories, test kit manufacturers, competent authorities, and food and feed business operators to implement microbiological methods.

Learn more about ISO 16140 series, and the necessary stages of validation and verification of methods before use.


Development of the ISO 16140 series

The ISO 16140 series consists now of six parts with the general title, Microbiology of the food chain - Method validation:

—    Part 1: Vocabulary;

—    Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method;

—    Part 3: Protocol for the verification of reference methods and validated alternative methods in a single laboratory;

—    Part 4: Protocol for method validation in a single laboratory;

—    Part 5: Protocol for factorial interlaboratory validation for non-proprietary methods;

—    Part 6: Protocol for the validation of alternative (proprietary) methods for microbiological confirmation and typing procedures.

The Introduction of parts 3, 4 and 5 provides a flow chart, in Figure 1, that gives an overview of the links between the different parts of the ISO 16140 series. It also guides the user in selecting the right part of the ISO 16140 series taking into account the purpose of the study.


ISO 17468 is a closely linked International Standard, which establishes technical rules for the development or revision and validation of standardized reference methods. More detailed information on ISO 17468 can be found here.


Validation of alternative versus reference methods: Part 2 of ISO 16140 series

Today, many alternative (mostly proprietary) methods exist that are used to assess the microbiological safety and quality of raw materials and finished food products and to monitor the microbiological status of manufacturing processes. The developers, end-users and public health authorities need a reliable common protocol for the validation of such alternative methods.

ISO 16140-2 is the base standard for alternative methods validation and is cross-referenced by the other parts of the 16140 series. It includes two phases, the method comparison study and the interlaboratory study, with separate protocols for the validation of qualitative and quantitative microbiological methods. The data generated will provide potential end-users with performance data for a given method, thus enabling them to make an informed choice on the implementation of a particular (alternative) method.

Data generated can also serve as a basis for the certification of an alternative method by an independent organization. The evaluation of the quality control of the alternative method’s manufacturing process is also an important aspect of this certification process. In the European Union, the validation and certification requirements for the use of alternative methods are included in the European Regulation 2073/2005.


From validation to verification

In general, two stages are needed before a method can be used in a laboratory: first, to prove that the method is fit for purpose and secondly, to demonstrate that the laboratory can properly perform the method.

—    The first stage is the validation of the method. Validation is conducted by performing a method comparison study, usually by one laboratory, followed by an interlaboratory study (see ISO 16140-2, ISO 16140-5 and ISO 16140-6). In the case when a method is validated within one laboratory only (see ISO 16140-4), no interlaboratory study is conducted.

—    The second stage is method verification, where a laboratory demonstrates that it can satisfactorily perform a validated method. This is described in ISO 16140-3 (publication planned in January 2021). Verification is only applicable to methods that have been validated using an interlaboratory study.


Different categories in the food chain

A category in the food chain is a group of sample types of the same origin, e.g. heat-processed milk and dairy products. An overview of categories is given in ISO 16140-2:2016, Annex A.

In a validation study, it is not feasible to test all existing categories of sample types; the diversity and number of samples used in any validation study is therefore limited. In most cases, the validation is based on 5 out of 15 defined food categories. Sometimes the validation is supplemented with other categories: pet food and animal feed, environmental samples (food or feed production), and/or primary production samples.

When a minimum of 5 different food categories are validated, the method is regarded as being validated for a “broad range of foods”; even though only 5 food categories are tested during the validation study, the method is expected to work for any type of food samples within the 15 food categories. In other words, the “scope” of validation of the method is a broad range of foods, corresponding to these 15 food categories.

The scope of validation is important for selecting categories, types and items for the method verification (according to ISO 16140-3), and to support this selection. Besides the scope of validation, the scope of the method and the scope of laboratory application are also of importance. The overlap between the different scopes, including an example, is illustrated in the Introduction of ISO 16140-3, Figure 3.


Part 3 of the ISO 16140 series: two stages of verification

As with validation, there are also two stages for verification of validated methods.

—    The first stage is named implementation verification. Its purpose is to demonstrate that the user laboratory can perform the method correctly. This is done by testing one of the same (food) items evaluated in the validation study – to demonstrate that the user can get a similar result – thereby affirming the user laboratory can perform the method correctly.

—    The second stage is named (food) item verification. Its purpose is to demonstrate that the user laboratory is capable of testing challenging (food) items generally tested within the laboratory’s scope of accreditation. This is accomplished by testing several of these (food) items and using defined performance characteristics to confirm the method performs well for these (food) items.


Transition period

At the time of publication of ISO 16140-3, some reference methods were not yet (fully) validated and would therefore fall outside the scope of this part of the ISO 16140 series. It is recognized that standardization organizations (including ISO and CEN committees) will need time to validate these reference methods. Therefore, these non-validated reference methods (including ISO or CEN standards) are verified in a user laboratory according to a specific protocol (see ISO 16140-3, Annex F). This is seen as a temporary situation until these methods are validated by the ISO and/or CEN committees. For further information, see the document: Transition period for the implementation of ISO 16140-3.


Part 4 and part 5 of the ISO 16140 series

ISO 16140-4 addresses validation within a single laboratory. The results are therefore only valid for the laboratory that conducted the study. In this case, verification (as described in ISO 16140-3) is not applicable. ISO 16140-5 describes protocols for non-proprietary methods in very specific cases, where a more rapid validation is required or when the method to be validated is highly specialized and the number of participating laboratories required by ISO 16140-2 cannot be reached.

ISO 16140-4 and ISO 16140-5 can be used for validation against a reference method. ISO 16140-4 (regarding qualitative and quantitative methods) and ISO 16140-5 (regarding quantitative methods only) can also be used for validation without a reference method.


Part 6 of the ISO 16140 series

ISO 16140-6 is somewhat different from the other parts in the ISO 16140 series in that it relates to a very specific situation where only the confirmation procedure of a method is to be validated independently to the detection or enumeration method [e.g. the biochemical confirmation of Enterobacteriaceae]. The confirmation procedure advances a suspected (presumptive) result to a confirmed positive result. The validation of alternative typing techniques (e.g. serotyping of Salmonella) is also covered by ISO 16140-6.

The validation study in ISO 16140-6 clearly defines the selective agar(s) from which strains can be confirmed using the alternative confirmation method. If successfully validated, the alternative confirmation method can only be used if strains are recovered on an agar that was used and shown to be acceptable within the validation study. Figure 2, in the Introduction of ISO 16140-6, shows the possibilities where an alternative confirmation method validated in accordance with ISO 16140-6 can be applied.


Supporting materials

To facilitate the implementation of the ISO 16140 series and especially ISO 16140-3:2021 ‘Method verification’, the following supporting materials are available:
  • Presentation: Overview of the ISO 16140 series – standards for validation and verification of microbiology methods (PDF or PowerPoint)
  • Presentation: Overview of ISO 16140-3 ‘Method verification’ – improving confidence in laboratory results (PDF or PowerPoint)
  • Presentation: “Deep dive” into ISO 16140-3 ‘Method verification’ – an extended training for improving confidence in laboratory results (PDF or PowerPoint)
  • Excel-calculation tool ISO 16140-3:2021 for assistance on statistics (link)
  • Recording of the Webinar on 2 March 2021: Publication ISO 16140-3 ‘Method verification’ (link)
Although the utmost care has been taken with the development of these supporting materials, errors and omissions cannot be entirely excluded. The Royal Netherlands Standardization Institute and/or the members of ISO/TC 34/SC 9/WG 3 ‘Method validation’ therefore accept no liability, not even for direct or indirect damage, occurring due to or in relation with the application of this presentation. In case there are differences between the content of these supporting materials and the International Standards, the content of the International Standards always prevails.‚Äč