Microbiology of the food chain - Method validation - Part 5: Protocol for factorial interlaboratory validation for non-proprietary methods (ISO 16140-5:2020)

The proposed deliverable specifies an alternative technical protocol for the validation of mostly non-proprietary methods in the field of microbiological analysis of food, animal feed, and environmental and primary production stage samples.
It is closely related to ISO 16140-2. The latter specifies the technical protocol for the validation of proprietary methods, including a classical interlaboratory study and a method comparison study to be conducted in one laboratory. The realization of classical interlaboratory studies demands a sufficient number of participating laboratories (at least 8 laboratories are required). There are many occasions where a sufficient number of participating laboratories is not available (e.g. when a new method is required quickly after an outbreak of a new microorganism). In this case, the validation cannot be considered as reliable any longer.
The proposed deliverable uses a modified protocol based on orthogonal, factorial studies. By selection of suitable influencing factors (technician, nutrient media, sample preparation, temperature, duration) a high certainty of the determined method validation parameters is obtained, so that the number of required collaborating laboratories can be reduced up to a minimum of 4.
This validation protocol can be used in different ways. If the 4 collaborators can be considered a “random sample” of independent and competent laboratories and from different organizations, the test method can be considered as being validated in the sense that accurate and precise measurements are to be expected from any competent laboratory. If the 4 collaborators can be considered a “random sample” of independent and competent laboratories from one organization, the test method can be considered as being validated in the sense that accurate and precise measurements are to be expected from any competent laboratory in this organization.

Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 5: Arbeitsvorschrift für eine faktorielle Ringversuch-Verfahrensvalidierung (ISO 16140-5:2020)

Dieses Dokument legt (auf der Grundlage von orthogonalen, faktoriellen Untersuchungen) die allgemeinen Grundsätze und technischen Arbeitsvorschriften für die Validierung nicht urheberrechtlich geschützter Verfahren in der Mikrobiologie der Lebensmittelkette fest.
Dieses Dokument ist anwendbar auf die Validierung von Verfahren zur Analyse (Nachweis oder Quantifizierung) von Mikroorganismen in:
- Erzeugnissen, die für den menschlichen Verzehr vorgesehen sind;
- Erzeugnissen, die als Futtermittel vorgesehen sind;
- Umgebungsproben im Bereich der Herstellung und Handhabung von Lebensmitteln und Futtermitteln;
- Proben aus dem Bereich der Primärproduktion.
Dieses Dokument ist insbesondere auf Bakterien und Pilze anwendbar. Einige Abschnitte können auf andere (Mikro )Organismen oder deren Metabolite angewendet werden. Die Bestimmung muss im Einzelfall erfolgen.
Dieses Dokument legt Arbeitsvorschriften für die Validierung mit Bezug auf ein Referenzverfahren sowohl für quantitative als auch qualitative Verfahren fest. Darüber hinaus stellt dieses Dokument eine Arbeitsvorschrift für die Validierung von quantitativen Verfahren ohne Bezug auf ein Referenzverfahren bereit. Qualitative Verfahren können nach diesem Dokument ohne Bezug auf ein Referenzverfahren nicht validiert werden.
ANMERKUNG ISO 16140 2 legt das allgemeine Prinzip und die technische Arbeitsvorschrift für die Validierung alternativer, zum Großteil urheberrechtlich geschützter Verfahren mit Bezug auf ein Referenzverfahren fest.
Dieses Dokument ist nur für die Validierung solcher Verfahren anwendbar, bei denen alle relevanten Parameter vollständig angegeben sind (einschließlich der Toleranzen zu Temperaturen und der Festlegungen zu Nährmedien) und die bereits optimiert wurden.
Verfahren, die nach diesem Dokument validiert wurden, können von den Laboren verwendet werden, die zu der festgelegten Grundgesamtheit der Labore gehören.

Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 5: Protocole pour la validation interlaboratoires de méthodes non commerciales par plan factoriel (ISO 16140-5:2020)

Le présent document établit les principes généraux ainsi que les protocoles techniques (fondé sur des études factorielles orthogonales) de validation de méthodes non commerciales, dans le domaine de la microbiologie de la chaîne alimentaire.
Le présent document est applicable à la validation de méthodes utilisées pour l'analyse (détection ou quantification) de micro-organismes présents dans:
—          les produits destinés à la consommation humaine;
—          les produits destinés à l'alimentation animale;
—          les échantillons environnementaux dans les domaines de la production et de la manutention de produits alimentaires;
—          les échantillons au stade de la production primaire.
Le présent document est notamment applicable aux bactéries et aux champignons. Certains articles peuvent être applicables à d'autres (micro-)organismes ou à leurs métabolites, qui doivent être déterminés au cas par cas.
Le présent document spécifie des protocoles pour la validation de méthodes quantitatives et qualitatives par rapport à une méthode de référence. Il établit également un protocole pour la validation de méthodes quantitatives sans méthode de référence. Les méthodes qualitatives ne peuvent pas être validées sans méthode de référence conformément au présent document.
NOTE       L'ISO 16140‑2 spécifie le principe général et le protocole technique pour la validation de méthodes alternatives, majoritairement commerciales, par rapport à une méthode de référence.
Le présent document est uniquement applicable à la validation de méthodes dont les paramètres pertinents sont intégralement spécifiés (notamment les tolérances sur les températures et les spécifications sur les milieux de culture) et qui ont déjà été optimisées.
Les méthodes qui ont été validées conformément au présent document peuvent être utilisées par les laboratoires de la population de laboratoires spécifiée.

Mikrobiologija v prehranski verigi - Validacija metode - 5. del: Protokol za medlaboratorijsko validacijo nelastniških metod (ISO 16140-5:2020)

General Information

Status
Published
Public Enquiry End Date
04-Mar-2018
Publication Date
08-Oct-2020
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
08-Sep-2020
Due Date
13-Nov-2020
Completion Date
09-Oct-2020

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Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 16140-5:2020
01-november-2020
Mikrobiologija v prehranski verigi - Validacija metode - 5. del: Protokol za
medlaboratorijsko validacijo nelastniških metod (ISO 16140-5:2020)
Microbiology of the food chain - Method validation - Part 5: Protocol for factorial
interlaboratory validation for non-proprietary methods (ISO 16140-5:2020)
Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 5: Arbeitsvorschrift für
eine faktorielle Ringversuch-Verfahrensvalidierung (ISO 16140-5:2020)
Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 5: Protocole
pour la validation interlaboratoires de méthodes non commerciales par plan factoriel
(ISO 16140-5:2020)
Ta slovenski standard je istoveten z: EN ISO 16140-5:2020
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 16140-5:2020 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 16140-5:2020

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SIST EN ISO 16140-5:2020


EN ISO 16140-5
EUROPEAN STANDARD

NORME EUROPÉENNE

August 2020
EUROPÄISCHE NORM
ICS 07.100.30
English Version

Microbiology of the food chain - Method validation - Part 5:
Protocol for factorial interlaboratory validation for non-
proprietary methods (ISO 16140-5:2020)
Microbiologie de la chaîne alimentaire - Validation des Mikrobiologie der Lebensmittelkette -
méthodes - Partie 5: Protocole pour la validation Verfahrensvalidierung - Teil 5: Arbeitsvorschrift für
interlaboratoires de méthodes non commerciales par eine faktorielle Ringversuch-Verfahrensvalidierung
plan factoriel (ISO 16140-5:2020) (ISO 16140-5:2020)
This European Standard was approved by CEN on 25 May 2020.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16140-5:2020 E
worldwide for CEN national Members.

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SIST EN ISO 16140-5:2020
EN ISO 16140-5:2020 (E)
Contents Page
European foreword . 3

2

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SIST EN ISO 16140-5:2020
EN ISO 16140-5:2020 (E)
European foreword
This document (EN ISO 16140-5:2020) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 463 “Microbiology of the food chain” the
secretariat of which is held by AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by February 2021, and conflicting national standards
shall be withdrawn at the latest by February 2021.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 16140-5:2020 has been approved by CEN as EN ISO 16140-5:2020 without any
modification.

3

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SIST EN ISO 16140-5:2020

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SIST EN ISO 16140-5:2020
INTERNATIONAL ISO
STANDARD 16140-5
First edition
2020-07
Microbiology of the food chain —
Method validation —
Part 5:
Protocol for factorial interlaboratory
validation for non-proprietary
methods
Microbiologie de la chaîne alimentaire — Validation des méthodes —
Partie 5: Protocole pour la validation interlaboratoires de méthodes
non commerciales par plan factoriel
Reference number
ISO 16140-5:2020(E)
©
ISO 2020

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SIST EN ISO 16140-5:2020
ISO 16140-5:2020(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2020
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved

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ISO 16140-5:2020(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 General principles for the factorial interlaboratory validation of non-proprietary
methods . 3
4.1 General . 3
4.2 Validation against a reference method . 3
4.3 Validation without a reference method . 3
5 Qualitative methods — Technical protocol for factorial interlaboratory validation .3
5.1 In-house validation study . 3
5.2 Interlaboratory validation study against a reference method . 4
5.2.1 General considerations . 4
5.2.2 Measurement protocol . 4
5.2.3 Selection of the factors to be studied . 5
5.2.4 Experimental design . 6
5.3 Calculations and summary of data . 6
5.4 Interpretation of data . 8
5.4.1 Paired study . 8
5.4.2 Unpaired study . 9
5.4.3 Analysis of factorial effects with respect to RLOD . 9
6 Quantitative methods — Technical protocol for factorial interlaboratory validation .10
6.1 In-house validation study .10
6.2 Interlaboratory validation study against a reference method .10
6.2.1 General considerations .10
6.2.2 Measurement protocol .10
6.2.3 Experimental design .11
6.3 Calculations, summary, and interpretation of data .12
6.3.1 Summary of test results .12
6.3.2 Precision data .13
6.3.3 Accuracy profile .14
6.4 Interlaboratory validation study without a reference method .14
Annex A (informative) List of factors and factor levels for factorial validation.15
Annex B (informative) Example of a factorial interlaboratory study for a quantitative method .17
Annex C (informative) Example of a factorial interlaboratory study for a qualitative method .28
Bibliography .34
© ISO 2020 – All rights reserved iii

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Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology, in collaboration with the European Committee for Standardization (CEN) Technical
Committee CEN/TC 463, Microbiology of the food chain, in accordance with the Agreement on technical
cooperation between ISO and CEN (Vienna Agreement).
A list of all parts in the ISO 16140 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2020 – All rights reserved

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SIST EN ISO 16140-5:2020
ISO 16140-5:2020(E)

Introduction
0.1  The ISO 16140 series
The ISO 16140 series has been expanded in response to the need for various ways to validate or verify
test methods. It is the successor to ISO 16140:2003. The ISO 16140 series consists 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.
ISO 17468 is a closely linked International Standard, which establishes technical rules for the
development and validation of standardized methods.
In general, two stages are needed before a method can be used in a laboratory.
— The first stage is the validation of the method. Validation is conducted using a study in a single
laboratory followed by an interlaboratory study (see ISO 16140-2, ISO 16140-6, and as described in
this document). In the case when a method is validated within one laboratory (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. Verification is only applicable to
methods that have been validated using an interlaboratory study.
In general, two types of methods are distinguished: reference methods and alternative methods.
A reference method is defined in ISO 16140-1:2016, 2.59, as an “internationally recognized and widely
accepted method”. The note to entry clarifies that “these are 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, 3.5, as a “reference method described in a standard”.
An alternative method (method submitted for validation) is defined in ISO 16140-1:2016, 2.4, 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”. The note to entry clarifies that:
“The method can be proprietary. The term ‘alternative’ is used to refer to the entire ‘test procedure
and reaction system’. This term includes all ingredients, whether material or otherwise, required for
implementing the method.”.
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. This document, ISO 16140-5, describes protocols for non-proprietary methods 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 this
document can be used for validation against a reference method. ISO 16140-4 (regarding qualitative
and quantitative methods) and this document (regarding quantitative methods only) can also be used
for validation without a reference method.
© ISO 2020 – All rights reserved v

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The flow chart in Figure 1 gives an overview of the links between the different parts mentioned above.
It also guides the user in selecting the right part of the ISO 16140 series, taking into account the purpose
of the study and the remarks given above.
Figure 1 — Flow chart for application of the ISO 16140 series
NOTE In this 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.
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 [e.g.
the biochemical confirmation of Enterobacteriaceae (see ISO 21528-2)]. 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 shows the possibilities where an alternative confirmation method validated
in accordance with ISO 16140-6 can be applied (see text in the boxes).
vi © ISO 2020 – All rights reserved

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SIST EN ISO 16140-5:2020
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Figure 2 — Use of validated alternative confirmation methods (see ISO 16140-6)
EXAMPLE An example application of a validated alternative confirmation method is as follows.
An alternative confirmation method based on ELISA has been validated (in accordance with ISO 16140-6) to
replace the biochemical confirmation for Salmonella as described in ISO 6579-1. In the validation study, XLD
(mandatory agar in accordance with ISO 6579-1) plus BGA and a specified chromogenic agar (two optional agars
for second plating in accordance with ISO 6579-1) were used as the agars to start the confirmation. The validated
confirmation method can be used to replace the biochemical confirmation under the following conditions:
— by laboratories using the ISO 6579-1; or
— by laboratories using an ISO 16140-2 validated alternative method that refers to ISO 6579-1 for confirmation; or
— by laboratories using an ISO 16140-2 validated alternative method that starts the confirmation from XLD
and/or BGA agar and/or the specified chromogenic agar.
The validated confirmation method cannot be used under the following conditions:
— by laboratories using an ISO 16140-2 validated alternative method that refers only to agars other than those
included in the validation to start the confirmation (e.g. Hektoen agar and SS agar only); or
— by laboratories using an ISO 16140-2 validated alternative method that refers only to a confirmation
procedure that does not require isolation on agar.
0.2  Validation protocols in the ISO 16140 series
An interlaboratory validation study, in accordance with ISO 16140-2, requires at least eight laboratories
for quantitative methods and at least ten laboratories for qualitative methods.
This document provides a protocol that addresses the special case where the number of laboratories
required in an interlaboratory validation of a method by ISO 16140-2 cannot be achieved. The protocol
allows a method validation based on a minimum of four laboratories. It applies, for example, in situations
where there is an urgent need for a validated method but the in-house and interlaboratory studies in
accordance with ISO 16140-2 take too long to complete. This document also addresses the problem of
method validation of highly specialized methods, for which only a few laboratories might be available
for a validation study. This document can only be used for non-proprietary methods. Table 1 provides
an overview of the different protocols.
© ISO 2020 – All rights reserved vii

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Table 1 — Overview of different validation protocols described in the ISO 16140 series
Number of participating
With reference method Without reference method
laboratories
1 ISO 16140-4 ISO 16140-4
4 to 7 (quantitative method)/ This document: for non-proprietary This document: for non-proprietary
4 to 9 (qualitative method) methods only quantitative methods only
≥ 8 (quantitative method)/ ISO 16140-2: for the interlaboratory
Not applicable
≥ 10 (qualitative method) study part
In order to reduce the number of required laboratories to a minimum of four, while maintaining the
applicability of the validation to all laboratories, the protocol is based on a factorial experimental design.
In the factorial design, factors such as the technician or culture medium are altered simultaneously,
and the method is used in a range of different factor settings. This approach allows a more detailed
analysis of the precision parameters of the method while, at the same time, requiring a smaller number
of laboratories and tests.
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SIST EN ISO 16140-5:2020
INTERNATIONAL STANDARD ISO 16140-5:2020(E)
Microbiology of the food chain — Method validation —
Part 5:
Protocol for factorial interlaboratory validation for non-
proprietary methods
1 Scope
This document specifies the general principles and the technical protocols (based on orthogonal,
factorial studies) for the validation of non-proprietary methods for microbiology of the food chain.
This document is applicable to the validation of methods used for the analysis (detection or
quantification) of microorganisms in:
— products intended for human consumption;
— products intended for animal feeding;
— environmental samples in the area of food and feed production, handling;
— samples from the primary production stage.
This document is, in particular, applicable to bacteria and fungi. Some clauses can be applicable to other
(micro)organisms or their metabolites, to be determined on a case-by-case basis.
This document specifies protocols for the validation against a reference method for both quantitative
and qualitative methods. This document also provides a protocol for the validation of quantitative
methods without a reference method. Qualitative methods cannot be validated without a reference
method in accordance with this document.
NOTE ISO 16140-2 specifies the general principle and the technical protocol for the validation of alternative,
mostly proprietary, methods against a reference method.
This document is only applicable to the validation of methods that are fully specified with regard to all
relevant parameters (including tolerances on temperatures and specifications on culture media) and
that have already been optimized.
Methods that have been validated in accordance with this document can be used by the laboratories of
the specified population of laboratories.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO 16140-1:2016, Microbiology of the food chain — Method validation — Part 1: Vocabulary
ISO 16140-2:2016, Microbiology of the food chain — Method validation — Part 2: Protocol for the validation
of alternative (proprietary) methods against a reference method
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3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 16140-1 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
factor
qualitative or quantitative parameter within the method that can be varied at two or more levels within
the limits of the specified method
EXAMPLE Technician.
Note 1 to entry: In this document, only those factors that are in line with the protocol of the method are
considered.
3.2
factor level
value of the factors (3.1) within the experimental design
EXAMPLE Technician “a”, technician “b”, etc.
Note 1 to entry: In this document, each factor is varied at two factor levels: “a” and “b”.
Note 2 to entry: This definition is based on how ISO 3534-3:2013, 3.1.12, defines “factor level”. In ISO 3534-3:2013,
3.1.12, the definition is more general, but the statistical meaning is the same.
3.3
orthogonal design
factorial design, in which for every pair of factors (3.1), each combination of factor levels (3.2) occurs the
same number of times across the possible factor levels
Note 1 to entry: This definition is based on how ISO 3534-3:2013, 3.1.31, defines “orthogonal array”, but for
“orthogonal design”, a more general and more theoretical definition is used.
3.4
reference value
estimated concentration level obtained with the reference method
3.5
reference value
estimated concentration
level obtained from the concentration level of the inoculum
3.6
setting
combination of factor levels (3.2)
EXAMPLE Technician “a” + culture media “b” + temperature “a” + etc.
Note 1 to entry: These conditions can be described by the combination of levels of factors varied within the study.
2 © ISO 2020 – All rights reserved

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4 General principles for the factorial interlaboratory validation of non-
proprietary methods
4.1 General
This document uses a protocol based on orthogonal, factorial studies. A high certainty of the determined
method validation parameters is achieved by focusing on suitable factors (e.g. technician, culture media,
sample preparation, temperature, test duration) that can influence the test
...

SLOVENSKI STANDARD
oSIST prEN ISO 16140-5:2018
01-marec-2018
Mikrobiologija v prehranski verigi - Validacija metode - 5. del: Protokol za
medlaboratorijsko validacijo nelastniških metod (ISO/DIS 16140-5:2017)
Microbiology of the food chain - Method validation - Part 5: Protocol for factorial
interlaboratory validation of non-proprietary methods (ISO/DIS 16140-5:2017)
Mikrobiologie von Lebensmitteln und Futtermitteln - Verfahrensvalidierung - Teil 5:
Arbeitsvorschrift für eine faktorielle Ringversuch-Verfahrensvalidierung (ISO/DIS 16140-
5:2017)
Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 5: Protocole
pour la validation interlaboratoires de méthodes non propriétaires par plan factoriel
(ISO/DIS 16140-5:2017)
Ta slovenski standard je istoveten z: prEN ISO 16140-5
ICS:
07.100.30 Mikrobiologija živil Food microbiology
oSIST prEN ISO 16140-5:2018 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 16140-5:2018

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oSIST prEN ISO 16140-5:2018
DRAFT INTERNATIONAL STANDARD
ISO/DIS 16140-5
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2017-12-15 2018-03-09
Microbiology of the food chain — Method validation —
Part 5:
Protocol for factorial interlaboratory validation of non-
proprietary methods
Microbiologie de la chaîne alimentaire — Validation des méthodes —
Partie 5: Protocole pour la validation interlaboratoires de méthodes non propriétaires par plan factoriel
ICS: 07.100.30
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
ISO/CEN PARALLEL PROCESSING
BEING ACCEPTABLE FOR INDUSTRIAL,
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STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
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WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 16140-5:2017(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
©
PROVIDE SUPPORTING DOCUMENTATION. ISO 2017

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oSIST prEN ISO 16140-5:2018
ISO/DIS 16140-5:2017(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2017, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
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ii © ISO 2017 – All rights reserved

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oSIST prEN ISO 16140-5:2018
ISO/DIS 16140-5:2017(E)
Contents
Foreword . 5
Introduction . 6
1 Scope . 9
2 Normative references . 9
3 Terms and definitions . 9
4 General principles for the factorial interlaboratory validation of non-proprietary methods . 10
4.1 General . 10
4.2 Validation against a reference method . 11
4.3 Validation without reference method . 11
5 Qualitative methods - Technical protocol for factorial interlaboratory validation . 11
5.1 In-house validation study . 11
5.2 Interlaboratory study . 12
5.2.1 General considerations . 12
5.2.2 Measurement protocol . 12
5.2.3 Experimental design . 14
5.3 Calculations and summary of data . 14
5.4 Interpretation of data . 16
5.4.1 Paired study . 16
5.4.2 Unpaired study . 16
5.4.3 Analysis of factorial effects . 17
6 Quantitative methods - Technical protocol for factorial interlaboratory validation. 17
6.1 In-house validation study . 17
6.2 Interlaboratory study . 18
6.2.1 General considerations . 18
6.2.3 Measurement protocol . 18
6.2.3 Experimental design . 19
6.3 Calculations and summary of data . 19
6.4 Precision study without a reference value . 22
Annex A (normative) — List of factors for factorial study design . 23
Annex B (informative) — Example of a factorial interlaboratory study for a quantitative method .
 . 25
B.1 Study design . 25
B.2 Calculations and summary of data . 27
B.2.1 Summary of the results of laboratories 1 to 5 of the interlaboratory study . 27
B.2.2 Precision data . 28
B.2.3 Accuracy profile . 30
Annex C (informative) — Example of a factorial interlaboratory study for a qualitative method .
 . 34
Bibliography . 42

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oSIST prEN ISO 16140-5:2018
ISO/DIS 16140-5:2017(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national
standards bodies (ISO member bodies). The work of preparing International Standards is normally
carried out through ISO technical committees. Each member body interested in a subject for which a
technical committee has been established has the right to be represented on that committee.
International organizations, governmental and non-governmental, in liaison with ISO, also take part in
the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all
matters of electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology (Working Group WG 3, Method validation).
A list of all parts of the ISO 16140 series can be found on the ISO website.


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Introduction
The ISO 16140 series has been elaborated in response to the need for various ways to validate or verify
test methods. It is the successor of ISO 16140:2003, Microbiology of food and animal feeding stuffs —
Protocol for the validation of alternative methods. ISO 16140 series consists of several 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 and validated alternative methods implemented in a
single laboratory
 Part 4: Protocol for single-laboratory (in-house) method validation
 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
ISO 17468, Microbiology of the food chain — Technical requirements and guidance on establishment or
[2]
revision of a standardized reference method , is a closely linked International Standard. This
International Standard, which establishes technical rules for the development and validation of
standardized methods, is intended for the development of standardized methods by ISO/TC 34, Food
products, Subcommittee SC 9, Microbiology and CEN/TC 275/WG 6, Microbiology of the food chain.
In general two stages are needed before a method can be used in a laboratory:
 The first stage is the validation of the method. This is either conducted in several laboratories
(parts 2 and 5 of ISO 16140) or in one laboratory (part 4 of ISO 16140).
 The second stage is method verification, where a laboratory demonstrates that it can satisfactorily
perform a validated method. This is described in part 3 of ISO 16140 (method verification). In
part 3, a separation is made between verification of (food) items that are included in the validation
study and (food) items that are not tested in the validation study but belong within the scope of
validation.
NOTE 1 Standardized reference methods (with and without published validation data) only require verification
before implementation in the laboratory.
NOTE 2 In this part of ISO 16140, the words ‘category’, ‘type’ and ‘item’ are sometimes combined with ‘food’ to
improve the readability of this document. However, the word ‘food’ is interchangeable with ‘feed’ and the other
areas of the food chain as mentioned in the Scope of ISO 16140-5.
Part 4 of ISO 16140 addresses validation within a single laboratory. The results are therefore only valid
in the laboratory which conducted the study. In this case, verification (part 3 of ISO 16140) is not
required.
Part 5 of ISO 16140 describes protocols for situations where a more rapid validation is required or
when the method to be validated is highly specialised, and, the number of participating laboratories
required by ISO 16140-2 cannot be reached.
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The flow chart in Figure 1 gives an overview of the links between the different parts mentioned above.
It also guides the users in selecting the right part of the ISO 16140 series, taking into account the
purpose of the study and the remarks given above. For this, it is important to distinguish between
'reference method' and 'standardized reference method'. A reference method is an internationally
recognized and widely accepted method (term 2.59 of ISO 16140-1:2016) and a standardized reference
method is a reference method described in a standard (term 3.5 of ISO 17468:2016). In the ISO 16140
series, reference method includes standardized reference method. The flow diagram acknowledges that
published validation data may not be available for some standardized reference methods.

Figure 1 — Flow chart for application of the different parts of the ISO 16140-series
Part 6 of ISO 16140, 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 validated. The
confirmation procedure advances a suspected (presumptive) result to a confirmed positive result. The
typing of pure strains (e.g. serotyping of Salmonella) is included in part 6 of ISO 16140.
An interlaboratory study, according to ISO 16140-2 (proprietary methods), requires at least
8 laboratories for quantitative methods and 10 laboratories for qualitative methods. ISO 16140-5 is
intended to be used for interlaboratory studies comprising 4-7 laboratories for quantitative methods
and 4-9 laboratories for qualitative methods. ISO 16140-5 can only be used for non-proprietary
methods. Table 1 provides an overview of the different protocols.

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oSIST prEN ISO 16140-5:2018
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Table 1 — Overview of different validation protocols described in ISO 16140
Number of laboratories With reference method Without reference method
1 Part 4 of ISO 16140: factorial or conventional Part 4 of ISO 16140: factorial or conventional
4 to 7 (quantitative method)/ Part 5 of ISO 16140: for non-proprietary Part 5 of ISO 16140: for non-proprietary
4 to 9 (qualitative method) methods only methods only
≥ 8 (quantitative method)/ Part 2 of ISO 16140 (for the interlaboratory Not available
≥ 10 (qualitative method) study part)

This part of ISO 16140 provides a protocol that addresses the special case where the number of
laboratories (8 for quantitative methods and 10 for qualitative methods) required in an interlaboratory
validation of a method by ISO 16140-2 cannot be achieved. The protocol allows a method validation
based on only 4 laboratories. It applies, for example, in situations where there is an urgent need for a
validated method but the protocols of ISO 16140-2 take too long to complete. This part of ISO 16140
also addresses the problem of method validation of highly specialised methods, for which only a few
laboratories might be available for a validation study.
In order to reduce the number of required laboratories to 4 while maintaining the generalizability of
the validation to all laboratories, the protocol is based on a factorial experimental design. In the
factorial design, several factors such as the technician or culture medium are altered simultaneously
and the method is used in a range of different factor settings. This approach allows a more detailed
analysis of the precision parameters of the method while, at the same time, requiring a smaller number
of laboratories and tests.


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oSIST prEN ISO 16140-5:2018
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Microbiology of the food chain — Method validation — Part 5:
Protocol for factorial interlaboratory validation of non-
proprietary methods
1 Scope
This part of ISO 16140 describes the general principle and the technical protocol (based on orthogonal,
factorial studies) for the validation of non-proprietary methods for microbiology of the food chain.
This part of ISO 16140 is applicable to the validation of non-proprietary methods used in the analysis
(detection or quantification) of microorganisms in
 products intended for human consumption,
 products intended for animal feeding,
 environmental samples in the area of food and feed production, handling, and
 samples from the primary production stage.
This part of ISO 16140 is in particular applicable to bacteria and fungi. Some clauses can be applicable
to other (micro)organisms or their metabolites, to be determined on a case-by-case-basis.
This part of ISO 16140 only applies to the validation of methods that are fully specified with regard to
all relevant parameters (including tolerances on temperatures and specifications on culture media) and
which have already been optimised.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO 16140-1:2016, Microbiology of the food chain — Method validation — Part 1: Vocabulary
ISO 16140-2:2016, Microbiology of the food chain — Method validation — Part 2: Protocol for the
validation of alternative (proprietary) methods against a reference method
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 16140-1 and the following
apply. ISO and IEC maintain terminological databases for use in standardization at the following
addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
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3.1
block
group of settings which have to be conducted in parallel or in a short time interval, and which are used
for the same samples
EXAMPLE Block = settings conducted in parallel =
Technician ‘a’ + culture medium ‘b’ + temperature ‘a’ + incubator ‘a’
AND
Technician ‘b’ + culture medium ‘a’ + temperature ‘b’ + incubator ‘b’

3.2
factor
qualitative or quantitative parameter within the method that can be varied at two or more levels within
the limits of the specified method
EXAMPLE Technician.
Note 1 to entry: In this part of ISO 16140, only those factors that are in line with the prescription of the method
are considered.
3.3
factor level
value of the factors within the experimental design
EXAMPLE Technician ‘a’, technician ‘b’, etc.
Note 1 to entry: In this part of ISO 16140, each factor is varied at two factor levels ‘a’ and ‘b’.
3.4
setting
combination of factor levels
EXAMPLE Technician ‘a’ + culture media ‘b’ + temperature ‘a’ + etc.
Note 1 to entry: These conditions can be described by the combination of levels of factors varied within the study.
4 General principles for the factorial interlaboratory validation of non-
proprietary methods
4.1 General
This part of ISO 16140 uses a protocol based on orthogonal, factorial studies. A high certainty of the
determined method validation parameters is obtained, by focussing on suitable factors (e.g. technician,
culture media, sample preparation, temperature, duration) that can influence the test result. This allows
the number of required laboratories to be reduced to a minimum of 4. General concepts and
considerations given in ISO 16140-2 shall be used, unless explicitly excluded. The validation can be
conducted against a reference method or by the use of appropriate reference materials.
This validation protocol can be used in different ways:
 For all laboratories: if 4 laboratories can be considered a ‘random sample’ of independent
laboratories from different organizations, the test method can be considered to be validated for all
laboratories in that accurate and precise measurements are to be expected from any laboratory.
 For laboratories within an organization: if 4 laboratories can be considered a ‘random sample’ of
independent laboratories from one organization, the test method can be considered to be validated
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for this organization in that accurate and precise measurements are to be expected from any
laboratory in the organization.
4.2 Validation against a reference method
The validation protocol comprises two phases:
 an in-house validation study of the non-proprietary method against the reference method carried
out in the organizing laboratory;
 an interlaboratory study of the non-proprietary method against the reference method carried out
in different laboratories.
The technical protocol for performing the in-house validation study and the interlaboratory study are
given in Clause 5 and 6, depending upon whether the test method is qualitative or quantitative in
nature.
The selected factors for the factorial interlaboratory study should be relevant to both the reference and
the alternative method.
4.3 Validation without reference method
The validation protocol comprises two phases:
 an in-house validation study of the non-proprietary method against the reference value carried out
in the organizing laboratory;
 an interlaboratory study of the non-proprietary method against the reference value carried out in
different laboratories.
The technical protocol for performing the in-house validation study and the interlaboratory study for
quantitative test methods are given in Clause 6.
Qualitative test methods cannot be validated without a reference method according to this part of
ISO 16140.
5 Qualitative methods - Technical protocol for factorial interlaboratory validation
5.1 In-house validation study
The in-house validation study is the part of the validation process that is performed in the organizing
laboratory. It can be conducted according to the conventional approach or the factorial approach, as
described in part 4 of ISO 16140.
An in-house validation study can be used in order to demonstrate the performance of the method for
the laboratory that conducted the study. In the framework of general method validation, it is the first
step and is needed for assessing the performance of the method across a large number of (food) types
and samples, whereas in the interlaboratory study, the performance of the method is assessed across a
larger number of laboratories.
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5.2 Interlaboratory study
5.2.1 General considerations
The aim of the interlaboratory study is to compare the performance of the reference method to the
alternative method in terms of the RLOD obtained by different laboratories, using the same set of
samples examined under reproducibility conditions and to compare these results with pre-set criteria
for the acceptable difference between the reference method and the alternative method. The
interlaboratory study is planned by the organizing laboratory.
The organizing laboratory prepares the samples and a data sheet for the recording of all experimental
data and critical experimental conditions used by each laboratory. It is necessary for each laboratory to
demonstrate its competence in the use of the alternative and the reference method according to
ISO 7218 prior to participating in the study.
Technicians, involved in the preparation of the samples used in the interlaboratory study, shall not take
part in the testing of the samples of the interlaboratory study.
5.2.2 Measurement protocol
Four or more independent laboratories are required to conduct the measurement series. These
laboratories shall be representative of the population of laboratories and shall belong to a minimum
of 3, but preferably 4 different organizations, excluding the organizing laboratory.
NOTE 1 Laboratories in different locations, but belonging to one company or institute, are accepted as different
organizations.
NOTE 2 If the 4 laboratories are independent and representative of the population of laboratories within one
organization, the outcome of the validation study applies only to laboratories within that organization.
If possible, more than 4 laboratories should participate, so that results from at least 8 technicians
remain even if, for some reason, certain data cannot be used.
The protocol is as follows:
 In cases where different enrichment protocols for the alternative method exist, the most
challenging enrichment protocol shall be selected, e.g. the protocol having the shortest incubation
time or the most selective enrichment conditions. The selected (food) item shall be relevant for the
chosen enrichment protocol. This relevant (food) type, which is used to prepare the test samples,
should contain a natural background microflora.
 The selected (food) item shall be inoculated with the target organism. The protocol for inoculation
of the samples shall be appropriate for the selected food. Samples shall be prepared by the
organizing laboratory to ensure homogeneity between and within samples using preparation
protocols contained in Annex B and Annex C of ISO 16140-2:2016.
 At least three different levels of contamination shall be used: a negative control (L ) and two levels
0
(L and L ). Level L should be between the LOD of the reference method (LOD ) and the LOD
1 2 1 50 50,ref 50
of the alternative method (LOD = RLOD · LOD ). Level L should be 1 log level above L .
50,alt 50,ref 2 10 1
 For the selected (food) item, for each setting and for each laboratory, 4 replicates shall be
conducted at level L Only 1 test is conducted at the two other levels L and L .
1. 0 2
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 According to the factorial, orthogonal design, 8 settings have to be conducted by each laboratory for
both test methods and for all levels of contamination (L , L and L ). In total 96 results
0 1 2
(1  4  1 tests × 8 settings × 2 methods) are conducted in each laboratory.
 All samples should b
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