SIST EN ISO 16140-2:2016

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Microbiology of the food chain - Method validation - Part 2: Protocol for the validation of
alternative (proprietary) methods against a reference method (ISO 16140-2:2016)
Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 2: Arbeitsvorschrift für
die Validierung von alternativen (urheberrechtlich geschützten) Verfahren anhand eines
Referenzverfahrens (ISO 16140-2:2016)
Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 2: Protocole
pour la validation de méthodes alternatives (commerciales) par rapport à une méthode
de référence (ISO 16140-2:2016)
Ta slovenski standard je istoveten z: EN ISO 16140-2:2016
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 16140-2
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2016
EUROPÄISCHE NORM
ICS 07.100.30 Supersedes EN ISO 16140:2003
English Version
Microbiology of the food chain - Method validation - Part 2:
Protocol for the validation of alternative (proprietary)
methods against a reference method (ISO 16140-2:2016)
Microbiologie de la chaîne alimentaire - Validation des Mikrobiologie der Lebensmittelkette -
méthodes - Partie 2: Protocole pour la validation de Verfahrensvalidierung - Teil 2: Arbeitsvorschrift für die
méthodes alternatives (commerciales) par rapport à Validierung von alternativen (urheberrechtlich
une méthode de référence (ISO 16140-2:2016) geschützten) Verfahren anhand eines
Referenzverfahrens (ISO 16140-2:2016)
This European Standard was approved by CEN on 12 May 2016.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, 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: Avenue Marnix 17, B-1000 Brussels
© 2016 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16140-2:2016 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
European foreword
This document (EN ISO 16140-2:2016) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”
the secretariat of which is held by DIN.
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 January 2017, and conflicting national standards shall
be withdrawn at the latest by January 2017.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent
rights.
This document supersedes EN ISO 16140:2003.
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, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 16140-2:2016 has been approved by CEN as EN ISO 16140-2:2016 without any
modification.
INTERNATIONAL ISO
STANDARD 16140-2
First edition
2016-06-15
Microbiology of the food chain —
Method validation —
Part 2:
Protocol for the validation of
alternative (proprietary) methods
against a reference method
Microbiologie de la chaîne alimentaire — Validation des méthodes —
Partie 2: Protocole pour la validation de méthodes alternatives
(commerciales) par rapport à une méthode de référence
Reference number
ISO 16140-2:2016(E)
©
ISO 2016
ISO 16140-2:2016(E)
© ISO 2016, Published in Switzerland
All rights reserved. Unless otherwise specified, 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.
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Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved

ISO 16140-2:2016(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General principles for the validation of alternative methods . 1
5 Qualitative methods — Technical protocol for validation . 2
5.1 Method comparison study . 2
5.1.1 General considerations . 2
5.1.2 Paired or unpaired study . 2
5.1.3 Sensitivity study . 2
5.1.4 Relative level of detection study . 7
5.1.5 Inclusivity and exclusivity study . 9
5.2 Interlaboratory study .10
5.2.1 General considerations .10
5.2.2 Measurement protocol .10
5.2.3 Calculations and summary of data .12
5.2.4 Interpretation of data .15
6 Quantitative methods — Technical protocol for validation .16
6.1 Method comparison study .16
6.1.1 General considerations .16
6.1.2 Relative trueness study .16
6.1.3 Accuracy profile study.20
6.1.4 Limit of quantification study .24
6.1.5 Inclusivity and exclusivity study .24
6.2 Interlaboratory study .26
6.2.1 General considerations .26
6.2.2 Measurement protocol .26
6.2.3 Calculations, summary, and interpretation of data .27
Annex A (informative) Classification of sample types and suggested target combinations for
validation studies .30
Annex B (normative) Order of preference for use of naturally and artificially contaminated
samples in validation studies .46
Annex C (informative) General protocols for contamination by mixture and artificial
contamination of foods .47
Annex D (informative) Models for RLOD calculations using data from the method
comparison study .50
Annex E (normative) Points to be considered when selecting strains for testing inclusivity
and exclusivity .52
Annex F (informative) Considerations for calculations of the relative level of detection
(RLOD) between laboratories as obtained in an interlaboratory study .54
Annex G (informative) Principle of the accuracy profile for validation of quantitative models .57
Annex H (informative) Application of the accuracy profile in the method comparison study .59
Annex I (informative) Example of the application of the accuracy profile for an
interlaboratory study .62
Bibliography .66
ISO 16140-2:2016(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 meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary Information
The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology.
This first edition of ISO 16140-2, together with ISO 16140-1, cancels and replaces ISO 16140:2003, which
has been technically revised. It also incorporates the Amendment ISO 16140:2003:Amd.1:2011.
ISO 16140 consists of the following parts, under 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
The following parts are under preparation:
— 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 of non-proprietary methods
— Part 6: Protocol for the validation of alternative (proprietary) methods for microbiological confirmation
and typing
iv © ISO 2016 – All rights reserved

ISO 16140-2:2016(E)
Introduction
Today, many alternative, mostly proprietary, methods exist that are used to assess the microbiological
quality of raw materials and finished products and the microbiological status of manufacturing
procedures. These methods are often faster and easier to perform than the corresponding standardized
method. The developers, end users, and authorities need a reliable common protocol for the validation
of such alternative methods. The data generated will also provide potential end users with performance
data for a given method, thus, enabling them to make an informed choice on the adoption of a particular
method. The data generated can also be the basis for the certification of a method by an independent
organization.
This part of ISO 16140
— is intended to provide a specific protocol and guidelines for the validation of proprietary
methods intended to be used as a rapid and/or easier method to perform than the corresponding
reference method,
— can also be used for the validation of other non-proprietary methods that are used instead of the
reference method,
— is intended as the successor of the validation protocol published in the first version of ISO 16140
(ISO 16140:2003), and
— is mainly written for the validation of methods that are capable of culturing the target microorganism,
but can also be applied to methods for microorganisms that cannot be cultured such as viruses (e.g.
Norovirus) and protozan parasites (e.g. Cryptosporidium or Giardia). In these cases, some wordings
are to be interpreted so as to fit the situation for non-culturable organisms.
The use of this part of ISO 16140 involves expertise on relevant areas such as microbiology, statistical
design, and analysis as indicated in the respective sections. The statistical expertise encompasses
overview of sampling theory and design of experiments, statistical analysis of (qualitative and
quantitative) microbiological data, and overview of statistical concepts on random sampling, sample
heterogeneity, sample stability, design of experiments, and variance components.
When this part of ISO 16140 is next reviewed, account will be taken of all information then available
regarding the extent to which the guidelines have been followed and the reasons for deviation from
them in the case of particular products.
The harmonization of validation methods cannot be immediate and for certain groups of products,
International Standards and/or national standards may already exist that do not comply with this part
of ISO 16140. It is hoped that when such standards are reviewed, they will be changed to comply with
ISO 16140 so that eventually, the only remaining departures from this part of ISO 16140 will be those
[3]
necessary for well-established technical reasons. For example, ISO 16297 deals with a very specific
validation for a specific subject (the hygienic status of raw milk samples) and will remain as a vertical
standard besides ISO 16140. If such a validation is needed, the vertical standard is more important.
INTERNATIONAL STANDARD ISO 16140-2:2016(E)
Microbiology of the food chain — Method validation —
Part 2:
Protocol for the validation of alternative (proprietary)
methods against a reference method
1 Scope
This part of ISO 16140 specifies the general principle and the technical protocol for the validation
of alternative, mostly proprietary, methods for microbiology in the food chain. Validation studies
according to this part of ISO 16140 are intended to be performed by organizations involved in method
validation.
This part of ISO 16140 is applicable to the validation of methods 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, and
— samples from the primary production stage.
This part of ISO 16140 is in particular applicable to bacteria and fungi. Some clauses of this part of
ISO 16140 could be applicable to other (micro) organisms or their metabolites on a case-by-case-basis.
In the future, guidance for other organisms (e.g. viruses and parasites) will be included in either this
part or a separate part of ISO 16140.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 16140-1, Microbiology of the food chain— Method validation — Part 1: Vocabulary
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 16140-1 apply.
4 General principles for the validation of alternative methods
The validation protocol comprises two phases:
— a method comparison study of the alternative (proprietary) method against the reference method
carried out in the organizing laboratory;
— an interlaboratory study of the alternative (proprietary) method against the reference method
carried out in different laboratories.
The technical rules for performing the method comparison study and the interlaboratory study are
given in Clause 5 and Clause 6, depending upon whether the alternative (proprietary) method is
ISO 16140-2:2016(E)
qualitative or quantitative in nature. The data generated in some parts of the validation study are
evaluated using the so-called Acceptability Limits (AL) and no statistical evaluation of the data are
conducted. These AL are based on experts’ opinion and data generated in existing validation studies.
5 Qualitative methods — Technical protocol for validation
5.1 Method comparison study
5.1.1 General considerations
The method comparison study is the part of the validation process that is performed in the organizing
laboratory. It consists of three parts namely the following:
— a comparative study of the results of the reference method to the results of the alternative method
in (naturally and/or artificially) contaminated samples (so-called sensitivity study);
— a comparative study to determine the relative level of detection (RLOD) in artificially contaminated
samples (so-called RLOD study);
— an inclusivity/exclusivity study of the alternative method.
The results (tables and calculations) of the different parts and the interpretation of the results, including
discrepant results, shall be given in a study report.
Test portions size shall be used as written in the reference method.
5.1.2 Paired or unpaired study
The reference and alternative methods shall be performed with, as far as possible, exactly the same
sample (same test portion). However, a distinction is made between studies where the same test portion
can be used for both the reference and the alternative method due to both methods having exactly the
same first step in the (enrichment) procedure and those where different test portions need to be used
for the reference and the alternative method (e.g. due to different enrichment broths). In the case where
the same test portion is used for both methods, the results from both methods are highly related to
each other. For example, when the sample is not contaminated, both methods should find the result of
that sample negative. Due to this relationship, the data produced by the reference and the alternative
method are named paired or matched. In this part of ISO 16140, the wording “paired study” will be
used for this type of study.
The opposite situation where there is no shared initial (enrichment) step for both the reference and
the alternative method is also possible. In this case, different test portions coming from the same batch
or lot of product have to be used for the two methods and the resulting data are named unpaired or
unmatched. In this part of ISO 16140, the word “unpaired study” will be used for this type of study. The
choice of having a paired study or an unpaired study depends on the protocols of the reference and
alternative method. If there is a common initial step in the (enrichment) procedures, a paired study
design is mandatory.
This clause describes the method comparison study if the reference and alternative method have a
joint initial step in the (enrichment) procedures (paired study) and if the reference and alternative
method do not have a joint initial (enrichment) step (unpaired study). Differences between both types
of studies are indicated in the text where appropriate.
5.1.3 Sensitivity study
The sensitivity study aims to determine the difference in sensitivity between the reference and the
alternative method. This study is conducted using naturally and/or artificially contaminated samples.
Different categories and types shall be tested for this. Acceptability Limits have been defined for the
2 © ISO 2016 – All rights reserved

ISO 16140-2:2016(E)
maximum acceptable difference depending on the type of study (paired/unpaired) and the number of
categories tested.
5.1.3.1 Selection of categories to be used
The selection of categories and types used within the validation will depend on the type or group of
microorganism and the scope of the validation.
If the method is to be applied for a broad range of foods, then at least five categories of food shall be
studied. The validation study report shall state the food categories used in the study. If the method
is to be validated for a restricted number of food categories, e.g. “ready-to-eat, ready-to-reheat meat
products”, and “heat-processed milk and dairy products”, then only these categories need to be studied.
In addition to food, feed samples, environmental samples, and primary production stage samples can be
included as additional categories. This will broaden the application of the use of the alternative method
for these additional categories.
For all selected categories (food and others), at least three different types per category shall be
included in the study. Annex A presents an overview of the relevant types and categories for specific
microorganisms that might be relevant for the validation. Annex A should be used to facilitate the
selection of categories, types, and items for the specific microorganism involved. It should not be
regarded as a mandatory choice.
When selecting samples for the study, it is of the highest priority to find those that are naturally
contaminated. If it is not possible to acquire a sufficient number of naturally contaminated samples,
artificial contamination of samples is permissible (see Annex B and Annex C). Details on the preparation
of the artificially inoculated samples should be given in the validation study report. It is desirable that
food samples come from as wide a distribution as possible in order to reduce any bias from local food
specialities and to broaden the range of validation.
It shall be ensured that with the selection of the different types, both high and low (natural) background
microflora, different types of stresses due to processing, and raw (unprocessed) items are included in
the study.
EXAMPLE For the validation of a method for detection of Listeria monocytogenes and the category “ready-
to-eat, ready-to-reheat meat products”, the types can be (1) cooked meat products (lower background flora, heat
stress), (2) fermented or dried meat products (high background flora, pH stress), and (3) raw cured (smoked)
(a <0,92) (intermediate background flora, a stress).
w w
In some cases, for example, for an alternative method that is applicable for a broad range of foods, it is
possible to combine the “ready-to-eat” and “raw” categories from the same product group. For example,
the categories raw and ready-to-eat meat (products) can be combined into one category having
three types divided over relevant raw and ready-to-eat food types. The selection of (combined) food
categories should be based on risk analysis.
5.1.3.2 Number of samples
For each category being examined, a minimum of 60 individual samples shall be tested made up of
at least three types with at least 20 samples representative for each type (three types × 20 samples
for each type = 60 samples). Fractional positive results by either the reference or alternative method
(i.e. samples should not be all positive or all negative) shall be obtained for each type tested. In the
ideal situation, 10 samples (50 %) tested per type should be positive and 10 negative, but should range
between 25 % and 75 %. For each category, at least 30 samples shall have a positive result by the
reference and/or the alternative method.
5.1.3.3 Alternative-method result and confirmation
Many alternative-method protocols contain two steps, the first being the enrichment and detection
step and the second being the confirmation of the detection result from step one. The end result
of the alternative method is the result after step two. The end result will be the same as the result
ISO 16140-2:2016(E)
after enrichment and detection in case there is no confirmation step included in the protocol of the
alternative method.
The (end) result of the alternative method shall be confirmed for the sensitivity study part. All results
obtained with the alternative method in an unpaired study shall be confirmed. In a paired study, only
the positive results obtained with the alternative method, for which the corresponding result with the
reference method was negative, shall be confirmed. This confirmation is needed to determine whether
the result is a true-positive or false-positive result. The confirmation test or tests shall be able to recover
and confirm the identity of the isolate as being the target of the method. These test(s) can be based on
the confirmation procedure of the reference method, the confirmation step of the alternative method
in case this procedure is able to isolate and confirm the identity of the target analyte, a combination of
both, or by any other means that is able to isolate and confirm the identity of the target analyte.
If the enrichments of the reference and alternative methods differ in terms of the number of enrichments
(i.e. primary/non-selective and secondary/selective) or total duration of incubation, an additional
confirmation pathway is necessary for the validation study. The first pathway shall be that to be used
with the alternative method according to its procedure/instructions (regular testing conditions by the
alternative method according to the kit insert procedure; this does not include the complementary tests
which can be performed during the validation study). The second pathway shall divert a portion of the
alternative method’s incubated enrichment to that of the reference method such that at minimum, the
total duration of incubation of the reference method enrichment(s) is/are respected. The results of the
two confirmation pathways are to be reported separately.
5.1.3.4 Calculation and interpretation for sensitivity
In general, the data shall be presented in a report in order to have an overview of the raw data
obtained. Information shall be given on the type of contamination (naturally contaminated or
artificially contaminated) of the samples used, the type of study design that was used (e.g. paired
study or unpaired study), and the confirmation test(s) used to confirm the alternative-method result.
For artificially contaminated samples, the (reference to the) procedure used for preparation shall be
specified (see also Annex C).
The results obtained for the reference and alternative methods originating from the same sample,
meaning from one test portion in case of a paired study or two test portions in case of an unpaired
study, shall be described for a paired study according to Table 1 and for an unpaired study according
to Table 2. Table 3 is prepared for the summarized sample results for all categories per category (≥60
samples) and per type (≥20 samples) for both a paired and unpaired study.
4 © ISO 2016 – All rights reserved

ISO 16140-2:2016(E)
Table 1 — Comparison and interpretation of sample results between the reference and
alternative methods for a paired study
Result of the (reference or alternative) method per sample
Reference Alternative method Confirmed Interpretation
method (including any confirmations alternative (based on the confirmed
as described in the method alternative-method result)
a
alternative-method protocol) (by any means)
b
+ + Not needed Positive Agreement (PA)
b
- - Not needed Negative Agreement (NA)
Negative Deviation due to false
b
+ - Not needed negative alternative-method result
(ND)
- + + Positive Deviation (PD)
Negative Agreement due to false
- + - positive alternative-method result
c
(NA)
a
Confirmation of the alternative-method result is done according to 5.1.3.3.
b
No need for additional confirmation test(s). Confirmed alternative-method result is the same as the alternative-
method result.
c
This false-positive result (FP) shall also be used to calculate the false positive ratio.
Table 2 — Comparison and interpretation of sample results between the reference and
alternative methods for an unpaired study
Result of the (reference or alternative) method per sample
Reference Alternative method Confirmed Interpretation
method (including any confirmations alternative method (based on the confirmed
a
as described in the (by any means) alternative-method result)
alternative-method protocol)
+ + + Positive Agreement (PA)
Negative Deviation due to false
+ + - positive alternative-method result
b
(ND)
- - - Negative Agreement (NA)
Negative Agreement due to false
- - + negative alternative-method result
(NA)
+ - - Negative Deviation (ND)
Negative Deviation due to false
+ - + negative alternative-method result
(ND)
- + + Positive Deviation (PD)
Negative Agreement due to false
- + - positive alternative-method result
b
(NA)
a
Confirmation of the alternative-method result is done according to 5.1.3.3
b
These false-positive results (FP) shall also be used to calculate the false positive ratio.
ISO 16140-2:2016(E)
Table 3 — Summary of results obtained with the reference and alternative methods of all
samples for each category
Reference-method positive Reference-method negative
(R+) (R-)
Alternative-method positive +/+ -/+
(A+) Positive Agreement (PA) Positive Deviation (PD)
Alternative-method negative +/- −/−
(A-) Negative Deviation (ND) Negative Agreement (NA)
Based on data summarized in Table 3 for the combined categories per category and per type, calculate
the values for sensitivity of the alternative method (1) and of the reference method (2), as well as the
relative trueness (3) and false positive ratio for the alternative method after the additional confirmation
of the results (4) as follows:
PA+PD
()
Sensitivity for the alternative method: SE = ×100% (1)
alt
()PA++ND PD
PA+ND
()
Sensitivity for the reference method: SE = ×100% (2)
ref
()PA++ND PD
PA +NA
()
Relative trueness: RT = ×100% (3)
N
FP
False positive ratio for the alternative method: FPR=×100% (4)
NA
where N is the total number of samples (NA + PA + PD + ND) and FP is the false-positive results. For
explanation of the abbreviations used, see Table 1 to Table 3.
The confirmed alternative-method results shall be used to determine whether the alternative method
produces comparable results to the reference method.
Calculate the difference between (ND – PD) for both paired and unpaired studies and the sum of
(ND + PD) for paired studies. Check whether the difference and/or sum of PD and ND conform to the
Acceptability Limit (AL) stated in Table 4 with respect to the type of study (paired or unpaired) and
the number of categories used in the evaluation.
NOTE Acceptability Limits (AL) are based on data and consensus expert opinion. The AL are not based on
statistical analysis of the data.
The interpretation of results shall be done per category and for all categories used in the validation
study. An interpretation of results shall also be done per enrichment protocol in case different
protocols are used for different types of samples. The AL is not met when the observed value is higher
than the AL. When the AL is not met, investigations should be made (e.g. root cause analysis) in order
to provide an explanation of the observed results. Based on the AL and the additional information, it is
decided whether the alternative method is regarded as not fit for purpose for the category or categories
involved. The reasons for acceptance of the alternative method in case the AL is not met shall be stated
in the study report.
6 © ISO 2016 – All rights reserved

ISO 16140-2:2016(E)
Table 4 — Acceptability limit parameters and values for a paired and unpaired study design in
relation to the number of categories used
Number of Paired study Unpaired study
categories
a b
(ND - PD ) (ND + PD) (ND - PD)
1 3 6 3
2 4 8 4
3 5 10 5
4 5 12 5
5 5 14 5
6 6 16 6
7 6 18 7
8 6 20 7
a
ND = number of samples with Negative Deviation results.
b
PD = number of samples with Positive Deviation results.
NOTE Information on differences observed between results of the alternative method before and after
confirmation of the results (step 1 and step 2) according to the alternative-method protocol should be presented
in the validation report as additional information, but is not used in the overall assessment of the alternative-
method performance.
5.1.4 Relative level of detection study
A comparative study is conducted to evaluate the level of detection (LOD) of the alternative method
against the reference method. The evaluation is based on the calculation of the relative level of detection
(RLOD). In the study, replicates of artificially contaminated samples are used at three or more levels of
contamination. Preferably, the levels are known as it allows calculation of the LOD. However, this is not
required.
5.1.4.1 Selection of categories, number of samples, and replicates tested
For the selection of categories and types, see 5.1.3.1. The same categories will be used as selected for
the sensitivity study (see 5.1.3). For each category, one relevant type is selected. In order to have a
better representation of the evaluated category, this type should be different from those used in
the sensitivity study (if possible). The samples shall be artificially inoculated. Procedures for the
preparation of artificially inoculated samples are presented in Annex C. Each type will be inoculated
with a different strain.
A minimum of three levels per type will be prepared consisting of at least a negative control level, a
low level, and a higher level. Ideally, the low level shall be the theoretical detection level (i.e. 0,7 cfu
per test portion) and the higher level just above the theoretical detection level (e.g. 1 cfu to 1,5 cfu per
test portion). At least the low level should have fractional recovery by the reference method (fractional
recovery at the low level should be between 25 % and 75 % of the number of samples tested). An
estimate for the level of contamination (except for the negative control) should be made. At the negative
control level, at least five replicate samples should be tested by both methods. For the second (low)
level (theoretical detection level), at least 20, and for the third (higher) level, at least five replicates
samples should be tested by both methods. The negative control level shall not produce positive results.
When positive results are obtained, the experiments have to be repeated for all levels.
Positive deviating test results obtained with the alternative method shall be additionally confirmed
(see 5.1.3.3). The RLOD shall be evaluated after confirmation.
NOTE 1 In order to have a better assurance that fractional recovery will be obtained, more levels of
contamination can be produced and tested.
NOTE 2 The level of contamination needed targets the LOD of the reference method if the alternative method
has a lower LOD than the reference method.
ISO 16140-2:2016(E)
5.1.4.2 Calculation and interpretation of the RLOD
The RLOD is defined as the ratio of the LODs of the alternative method and the reference method:
LOD
alt
RLOD= (5)
LOD
ref
For each category, at least the RLODs shall be estimated by fitting a complementary-log-log (CLL) model
to the combined absence/presence data of both methods as a function of method. The contamination
levels are not needed for the calculations of the RLOD since they are included in the model resulting in
curves in a graph of probability of detection versus log dose (contamination level). The statistical model
1) ®
and the calculations are worked out in Annex D. Calculations can be performed with the Excel ®
spreadsheet of this part of ISO 16140. The Excel spreadsheet for calculating RLOD values is freely
available for download at http://standards.iso.org/iso/16140 and then select the RLOD file. For ®
calculations using this Excel spreadsheet, the option of “unknown concentration” shall be used.
Calculate for each item i theRLOD . Tabulate the results as indicated in Table 5.
i
Table 5 — Presentation of RLOD before and after confirmation of the alternative-method results
RLOD using the RLOD using the confirmed

alternative-method results alternative-method results
Item
(category) RLOD RLOD
i i
(i)
…
k
Combined
An Acceptability Limit (AL) for the RLOD based on the confirmed alternative-method results specifies
the maximum increase in LOD of the alternative versus the reference method that would not be
considered as relevant in consideration of the fitness for purpose of the method. Consequently, AL will
be a value >1. The interpretation should be made for each item.
The AL for paired study data are set at 1,5, meaning that the LOD for the alternative method shall not be
higher than 1,5 times the LOD of the reference method. An LOD value for the alternative method smaller
than the LOD value for the reference method is always accepted as this means that the alternative
method is likely to detect lower levels of contamination than the reference method.
The AL for unpaired study data are set at 2,5, meaning that the LOD for the alternative method
shall not be higher than 2,5 times the LOD of the reference method. An LOD value for the alternativ
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