Mastering Method Validation: Navigating the ISO 16140 Series for Food Microbiology Method Validation

Introduction

Welcome, fellow laboratory professionals! We are excited to share insights about the ISO 16140 series - a critical framework for method validation in food microbiology. This series has evolved significantly in response to the growing need for various ways to validate and verify test methods, replacing the previous ISO 16140:2003 standard.

The ISO 16140 series consists of six interconnected parts, each addressing specific aspects of method validation in the microbiology of the food chain:

1. Part 1: Vocabulary
2. Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method
3. Part 3: Protocol for the verification of reference methods and validated alternative methods in a single laboratory
4. Part 4: Protocol for method validation in a single laboratory
5. Part 5: Protocol for factorial interlaboratory validation for non-proprietary methods
6. Part 6: Protocol for the validation of alternative (proprietary) methods for microbiological confirmation and typing procedures

Additionally, ISO 17468 is closely linked to this series, establishing technical rules for the development and validation of standardized methods.

Two Essential Stages Before Method Implementation

Before any method can be used in a laboratory, two critical stages must typically be completed:

Stage 1: Method Validation

Validation confirms that a method is fit for its intended purpose. This can be conducted in two ways:

• Multi-laboratory validation: Involves a study in a single laboratory followed by an interlaboratory study (as described in ISO 16140-2, ISO 16140-5, and ISO 16140-6)
• Single laboratory validation: When a method is validated within one laboratory only (as described in ISO 16140-4), no interlaboratory study is conducted

Stage 2: Method Verification

Verification is where a laboratory demonstrates that it can satisfactorily perform a validated method. This is described in ISO 16140-3 and is only applicable to methods that have been validated using an interlaboratory study.

Understanding Method Types

The ISO 16140 series distinguishes between two primary types of methods:

Reference Methods

A reference method is defined as an "internationally recognized and widely accepted method." These typically include ISO standards and standards jointly published by ISO and CEN or other regional/national standards of equivalent standing. In the ISO 16140 series, reference methods include standardized reference (ISO and CEN) methods as defined in ISO 17468:2016 as a "reference method described in a standard."

Alternative Methods

An alternative method is defined as a "method of analysis that detects or quantifies, for a given category of products, the same analyte as is detected or quantified using the corresponding reference method."

Important note: The term 'alternative' refers to the entire 'test procedure and reaction system' and includes all ingredients, whether material or otherwise, required for implementing the method. These methods can be proprietary.

Focus on ISO 16140-4: Single Laboratory Validation

ISO 16140-4 specifically addresses validation within a single laboratory. The key distinction here is that the results are only valid for the laboratory that conducted the study. In this case, verification (as described in ISO 16140-3) is not applicable.

This document can be used for:

• Validation against a reference method
• Validation without a reference method (for both qualitative and quantitative methods)

Similarly, ISO 16140-5 can be used for:

• Validation against a reference method
• Validation without a reference method (but only for quantitative methods)

Navigating the ISO 16140 Series: The Flow Chart Guide

The flow chart in Figure 1 provides an excellent visual guide to help you select the right part of the ISO 16140 series based on your specific needs. It takes into account the purpose of your study and other relevant factors.

How to Use the Flow Chart:

1. Start by determining if your method is already validated (with performance characteristics provided)
2. If not validated, determine your validation needs:
   • For alternative (proprietary) methods → Choose ISO 16140-2
   • For non-proprietary methods → Choose ISO 16140-5
   • For single-laboratory validation → Choose ISO 16140-4
   • For reference methods → Choose ISO 17468
3. If the method is validated, check if it's validated according to ISO 16140-4
   • If yes, apply the method only in that particular laboratory (including scope extension)
   • If no, determine if the food category to be tested is in the scope of the method
4. If the food category is not in the scope, choose the appropriate standard for scope extension
5. If the food category is in the scope, choose ISO 16140-3 for verification

Important Terminology Note

It's worth highlighting that in the ISO 16140-4 document, the words "category", "type" and/or "item" are sometimes combined with "(food)" to improve readability. However, the word "(food)" is interchangeable with "(feed)" and other areas of the food chain as mentioned in Clause 1. This flexibility allows the standard to be applied across various sectors of the food chain.

Understanding ISO 16140-6: Alternative Confirmation Methods

ISO 16140-6 stands somewhat apart from the other parts in the series as it addresses a very specific situation: the validation of confirmation procedures or typing techniques.

For example, it covers:

• The biochemical confirmation of Enterobacteriaceae (see ISO 21528-2)
• Alternative typing techniques (e.g., serotyping of Salmonella)

The confirmation procedure advances a suspected (presumptive) result to a confirmed positive result.

Limitations of Validated Confirmation Methods

The validation study in ISO 16140-6 clearly defines the selective agar(s) from which strains can be confirmed using the alternative confirmation method. An important restriction: 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.

This means the validated confirmation method cannot be used when:

• Strains are recovered on agars that were not included in the validation study
• The recovery conditions differ from those specified in the validation study
• The target organisms are outside the scope defined in the validation

Practical Application Example

Let's consider a practical example to illustrate how this works:

Scenario: Your laboratory needs to implement a new method for detecting Salmonella in ready-to-eat foods.

Decision Process:

1. Is the method already validated? No.
2. What type of method is it? It's a proprietary rapid method.
3. According to the flow chart, you should use ISO 16140-2 for validation.
4. After validation, when implementing in your laboratory, you would use ISO 16140-3 for verification.

If instead you were developing your own in-house method for a specific food matrix not covered by existing methods, you would use ISO 16140-4 for single-laboratory validation.

Conclusion

The ISO 16140 series provides a comprehensive framework for method validation in food microbiology, with each part addressing specific validation scenarios. Understanding the relationships between these parts is crucial for selecting the appropriate validation approach for your laboratory's needs.

For those working in ISO 17025 accredited laboratories, proper method validation and verification are essential components of demonstrating technical competence. The ISO 16140 series offers structured, internationally recognized protocols to achieve this.

I hope this overview helps you navigate the ISO 16140 series more effectively! Feel free to share your experiences or questions about implementing these standards in your laboratory.