ISO 17822:2020
(Main)In vitro diagnostic test systems — Nucleic acid amplification-based examination procedures for detection and identification of microbial pathogens — Laboratory quality practice guide
In vitro diagnostic test systems — Nucleic acid amplification-based examination procedures for detection and identification of microbial pathogens — Laboratory quality practice guide
This document describes the particular clinical laboratory practice requirements to ensure the quality of detection, identification and quantification of microbial pathogens using nucleic acid amplification tests (NAAT). It is intended for use by laboratories that develop, and/or implement and use, or perform NAAT for medical, research or health-related purposes. This document does not apply to the development of in vitro diagnostic (IVD) medical devices by manufacturers. However, it does include verification and validation of such devices and/or the corresponding processes when implemented and used by the laboratories.
Systèmes d'essai pour diagnostic in vitro — Modes opératoires d'examen in vitro qualitatifs fondés sur l'acide nucléique pour la détection et l'identification d'agents pathogènes microbiens — Guide pratique sur la qualité dans les laboratoires
General Information
Relations
Buy Standard
Standards Content (Sample)
INTERNATIONAL ISO
STANDARD 17822
First edition
2020-11
In vitro diagnostic test systems —
Nucleic acid amplification-based
examination procedures for detection
and identification of microbial
pathogens - Laboratory quality
practice guide —
Part :
Laboratory quality practice guide
Systèmes d'essai pour diagnostic in vitro — Modes opératoires
d'examen in vitro qualitatifs fondés sur l'acide nucléique pour la
détection et l'identification d'agents pathogènes microbiens — Guide
pratique sur la qualité dans les laboratoires —
Partie : Guide pratique de qualité au laboratoire
Reference number
ISO 17822:2020(E)
©
ISO 2020
---------------------- Page: 1 ----------------------
ISO 17822:2020(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
---------------------- Page: 2 ----------------------
ISO 17822:2020(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General laboratory requirement for microbial pathogens NAAT .10
4.1 General laboratory risk management and biosafety requirements .10
4.2 General laboratory set ups for pathogen detection .10
4.3 Commercial equipment (including software programs) .12
4.4 Laboratory personnel .12
5 Planning and implementation of pathogen NAAT assay .12
5.1 Quality control material .13
5.1.1 Examination of quality control material .13
5.1.2 Defining target sequence .15
5.2 Verification and validation .16
6 Verification or validation of test systems .16
6.1 Predicate assay selection by method comparison.16
7 Assay design and development of LDT .17
7.1 General .17
7.1.1 Definition of customer/patient’s and stakeholder needs of the intended
use of the assay .17
7.1.2 General criteria for Verification of assay .18
7.1.3 Specific criteria for verification of assay design input specifications .18
7.1.4 Validation of intended use .19
7.2 Diagnostic workflow analysis for Nucleic NAAT procedure .19
7.2.1 Pre-analytical workflow requirements .19
7.2.2 Analytical workflow requirements .20
7.2.3 Post-analytical workflow requirements .22
7.3 Verification and validation performance characteristics .23
7.3.1 Range of detection .23
7.3.2 Test accuracy (Trueness and Precision) .23
7.4 Analytical sensitivity / limit of detection .25
7.4.1 Validation of assay .26
8 Implementation and use in the laboratory .27
9 Reporting and interpretation of results .27
10 Quality assurance procedures .28
10.1 Performance monitoring and optimization of the assay .28
10.2 Inter-laboratory comparison .29
Annex A (informative) Pre-analytical consideration for sample preparation .30
Annex B (informative) Verification and validation of assays .37
Bibliography .38
© ISO 2020 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO 17822:2020(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 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 212, Clinical laboratory testing and in
vitro diagnostic test systems.
This first edition of ISO 17822 cancels and replaces ISO/TS 17822-1:2014, which has been technically
revised. The main changes are as follows:
— Clause 4 has been updated and merged from ISO/TS 17822-1:2014.
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
---------------------- Page: 4 ----------------------
ISO 17822:2020(E)
Introduction
Nucleic acid amplification-based tests (NAATs) are now commonly used in in vitro diagnostic (IVD)
tools in laboratory medicine for the detection, identification and quantification of microbial pathogens.
The NAAT result is influenced by all steps of the entire diagnostic workflow (i.e. pre-examination,
examination, post-examination). Therefore, this document considers all critical aspects of the entire
diagnostic workflow when designing, developing and implementing and using a specific microbial
pathogen NAAT examination.
NAAT examinations include PCR technology as well as other amplification-based technologies such
as, but not limited to, loop-mediated isothermal amplification (LAMP), transcription-mediated
amplification (TMA) and strand displacement amplification (SDA).
This document covers the implementation of commercially available IVD(s) into the medical laboratory
routine use as well as the design, development and implementation of laboratory developed tests (LDT).
This document will address the additional specific considerations, requirements and recommendations
for the detection of microbial pathogens with sampling, nucleic acid extraction, genetic heterogeneity
and the laboratory containment category which is required.
Due to high analytical sensitivity of nucleic acid-based examination procedures, special attention to
their design, development and use is required. This includes verification of analytical and validation of
clinical performance characteristics.
In this document, the following verbal forms are used:
— “shall” indicates a requirement;
— “should” indicates a recommendation;
— “may” indicates a permission;
— “can” indicates a possibility or a capability.
© ISO 2020 – All rights reserved v
---------------------- Page: 5 ----------------------
INTERNATIONAL STANDARD ISO 17822:2020(E)
In vitro diagnostic test systems — Nucleic acid
amplification-based examination procedures for detection
and identification of microbial pathogens - Laboratory
quality practice guide —
Part :
Laboratory quality practice guide
1 Scope
This document describes the particular clinical laboratory practice requirements to ensure the quality
of detection, identification and quantification of microbial pathogens using nucleic acid amplification
tests (NAAT).
It is intended for use by laboratories that develop, and/or implement and use, or perform NAAT for
medical, research or health-related purposes. This document does not apply to the development of in
vitro diagnostic (IVD) medical devices by manufacturers. However, it does include verification and
validation of such devices and/or the corresponding processes when implemented and used by the
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 15189, Medical laboratories — Requirements for quality and competence
ISO 15190, Medical laboratories — Requirements for safety
3 Terms and definitions
For the purposes of this document, the following terms and definitions 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
accuracy
closeness of agreement between a test result or measurement result and the true value
Note 1 to entry: In practice, the accepted reference value is substituted for the true value.
Note 2 to entry: The term “accuracy”, when applied to a set of test or measurement results, involves a combination
of random components and a common systematic error or bias component.
Note 3 to entry: Accuracy refers to a combination of trueness and precision.
[SOURCE: ISO 3534-2:2006]
© ISO 2020 – All rights reserved 1
---------------------- Page: 6 ----------------------
ISO 17822:2020(E)
3.2
amplification product
amplicon
specific DNA (3.17) fragment produced by a DNA-amplification technology, such as the polymerase chain
reaction (PCR) (3.34)
[SOURCE: ISO 13495:2013, 3.3.1]
3.3
analytical specificity
specificity
capability of a measuring system, using a specified measurement procedure, to provide measurement
results for one or more measurands (3.28) which do not depend on each other nor on any other quantity
in the system undergoing measurement
[21]
Note 1 to entry: Lack of analytical specificity is called analytical interference (see ISO 18113-1:2009, A.3.2 ).
Note 2 to entry: Specificity of a measurement procedure should not be confused with clinical specificity (SOURCE:
[21]
ISO 18113-1:2009, A.3.16 ).
[22]
Note 3 to entry: VIM; JCGM 200; 2012 uses the term selectivity for this concept instead of specificity.
Note 4 to entry: For qualitative and semiquantitative examination procedures, analytical specificity is determined
by the ability to obtain negative results in concordance with negative results obtained by the reference method.
[SOURCE: ISO 18113-1:2009, A.3.4]
3.4
biorisk
probability or chance that a particular adverse event (in the context of this document: accidental
infection or unauthorized access, loss, theft, misuse, diversion or intentional release), possibly leading
to harm, will occur
[SOURCE: WHO Biorisk management , Laboratory biosecurity guidance , September 2006]
3.5
biosafety
describes the containment principles, technologies and practices that are implemented to prevent the
unintentional exposure to pathogens and toxins, or their accidental release
[SOURCE: WHO Biorisk management Laboratory biosecurity guidance September 2006]
3.6
biosecurity
set of preventive measures and actions to reduce the risk of intentional or unintentional transmission
of infectious diseases
Note 1 to entry: Biosecurity encompasses the prevention of the intentional removal (theft) of biological materials
from laboratories.
Note 2 to entry: These preventive measures are a combination of systems and practices implemented in
laboratories against the use of dangerous pathogens and toxins for malicious use to prevent the spread of these
biological agents.
2 © ISO 2020 – All rights reserved
---------------------- Page: 7 ----------------------
ISO 17822:2020(E)
3.7
calibration
operation that, under specified conditions, in a first step, established a relation between the quantity
values with measurement uncertainties provided by measurement standards and corresponding
indications with associated measurement uncertainties and, in second step, uses this information to
establish a relation for obtaining a measurement result from an indication
Note 1 to entry: according to US Code of Federal Regulations, calibration is a process of testing and adjusting an
instrument or test system to establish a correlation between the measurement response and the concentration
[20]
or amount of the substance that is being measured by the test procedure (modified from 42CFR 493.1218) .
[SOURCE: VIM; JCGM 200; 2012]
3.8
certified reference material
CRM
reference material (RM) (3.41) characterized by a metrologically valid procedure for one or more
specified properties, accompanied by a RM (3.41) certificate that provides the value of the specified
property, its associated uncertainty, and a statement of metrological traceability
Note 1 to entry: The concept of value includes a nominal property or a qualitative attribute such as identity or
sequence. Uncertainties for such attributes may be expressed as probabilities or levels of confidence.
Note 2 to entry: Metrologically valid procedures for the production and certification of RMs (3.41) are given in,
[17]
among others, ISO 17034 .
[19]
Note 3 to entry: ISO/IEC Guide 99:2007 has an analogous definition.
[SOURCE: ISO Guide 30, 2.1.2]
3.9
clinical performance
ability of an in vitro diagnostic examination procedure to yield results that
are correlated with a specific clinical condition or physiological state in accordance with the target
population and intended user
Note 1 to entry: Although sometimes referred to as diagnostic performance or clinical validity; clinical
performance is the harmonized term endorsed by the Global Harmonization Task Force (GHTF) and its successor,
the International Medical Devices Regulators Forum (IMDRF).
Note 2 to entry: Evaluation of clinical performance often relies on the outcome of other types of clinical
examinations to define "true positive or true negative" results.
[SOURCE: GHTF/ SG5/N 6:2012, 4.4.2, modified — medical device has been changed to — examination
procedure and particularly has been changed to — specific.]
3.10
clinical sensitivity
diagnostic sensitivity
ability of an in vitro diagnostic examination procedure to identify the presence
of a target marker associated with a specific disease or condition
Note 1 to entry: Also defined as percent positivity in samples (3.44) where the target marker is known to be
present.
Note 2 to entry: Diagnostic sensitivity is expressed as a percentage (number fraction multiplied by 100),
calculated as 100 × the number of true positive values (TP) divided by the sum of the number of true positive
values (TP) plus the number of false negative values (FP), or 100 × TP/(TP + FN). This calculation is based on a
study design where only one sample (3.44) is taken from each subject.
Note 3 to entry: The target condition is defined by criteria independent of the examination procedure under
consideration.
© ISO 2020 – All rights reserved 3
---------------------- Page: 8 ----------------------
ISO 17822:2020(E)
[SOURCE: ISO 18113-1:2009, A.3.15]
3.11
clinical specificity
diagnostic specificity
ability of an in vitro diagnostic examination procedure to recognise the absence
of a target marker associated with a specific disease or condition
Note 1 to entry: Also defined as percent negativity in samples (3.44) where the target marker is known to be absent.
Note 2 to entry: Clinical specificity is expressed as a percentage (number fraction multiplied by 100), calculated
as 100× the number of true negative values (TN) divided by the sum of the number of true negative plus the
number of false positive (FP) values, or 100 × TN/(TN + FP). This calculation is based on a study design where
only one sample (3.44) is taken from each subject.
Note 3 to entry: The target condition is defined by criteria independent of the examination procedure under
consideration.
[SOURCE: ISO 18113-1:2009, A.3.16]
3.12
complementary DNA
cDNA
single-stranded DNA (3.17) that is complementary to a given RNA (3.42) and synthesized in the presence
of reverse transcriptase to serve as a template (3.47) for synthesis of DNA (3.17) copies
3.13
contamination
introduction of an undesirable substance or matter
3.14
cut-off value
quantity value used as a limit to identify samples (3.44) that indicate the presence or the absence of a
specific disease, condition or measurand (3.28)
Note 1 to entry: Defines which measurement results are reported as positive and which are reported as negative.
Note 2 to entry: Measurement results near the cut-off value can be considered inconclusive.
Note 3 to entry: The selection of the cut-off value determines the clinical specificity (3.11) and clinical sensitivity
(3.10) of the examination.
3.15
denaturation
physical and/or chemical treatment which results in the separation of nucleic acid double helices
Note 1 to entry: denaturation of DNA (3.17) results in separation of double-stranded DNA (3.17) into single-
stranded DNA (3.17).
[SOURCE: ISO 21572:2013, 3.1.6 — modified, term ”denaturation of proteins” has been changed to
”denaturation”, and ” the POI or” has been deleted. Note 1 to entry has been added.]
3.16
deoxyribonucleoside triphosphate
dNTP
solution containing dATP, dCTP, dGTP, dTTP and/or dUTP
[SOURCE: ISO 22174:2005, 3.3.7]
4 © ISO 2020 – All rights reserved
---------------------- Page: 9 ----------------------
ISO 17822:2020(E)
3.17
DNA
deoxyribonucleic acid
polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded
(ssDNA) form
[SOURCE: ISO 22174:2005, 3.1.2]
3.18
DNA polymerase for PCR
thermostable enzyme which catalyses repeated DNA (3.17) synthesis
[SOURCE: ISO 22174:2005, 3.4.17]
3.19
DNA sequencing
determining the order of nucleotide bases (adenine, guanine, cytosine, and thymine) in a molecule of
DNA (3.17)
Note 1 to entry: Sequence is generally described from the 5’ end.
3.20
hybridization
specific binding of complementary nucleic acid (3.32) sequences under suitable reaction conditions
[SOURCE: ISO 22174:2005, 3.6.3]
3.21
inhibition
reduction of amplification or interference with detection process that can lead to false negative results
or reduced quantity
3.22
interfering substances
endogenous or exogenous substances in clinical specimens/samples (3.44) that can alter
an examination result
[SOURCE: ISO 20186:2019-1, 3.15 modified]
3.23
inhouse assay
laboratory developed test
LDT
type of in vitro diagnostic test that is designed, manufactured and used within a single laboratory
Note 1 to entry: Inhouse assay/LDT needs to be validated for its intended use before putting into service.
3.24
linearity
ability of a method of analysis, within a certain range, to provide an instrumental response or results
proportional to the quantity of nucleic acid target sequence (3.46) to be determinded in the laboratory
sample (3.44)
Note 1 to entry: In the case of qPCR, the quantification cycle (also termed cycle threshold or crossing point) is
inversely proportional to the quantity of nucleic acid target sequence. (3.46).
Note 2 to entry: The term linearity is frequently linked with the linear range of the method and refers to the
ability of a method to give a response or result that is directly propotional to the concentration of the nucleic acid
target sequence (3.46).
[SOURCE: ISO 16577:2016, 3.92 modified — Notes 1 and 2 to entry added; ‘quantity of analyte’ replaced
with ‘quantity of the nucleic acid target sequence’ (3.46).]
© ISO 2020 – All rights reserved 5
---------------------- Page: 10 ----------------------
ISO 17822:2020(E)
3.25
limit of detection
LOD
measured quantity value, obtained by a given measurement procedure, for which the probability of
falsely claiming the absence of a component in a material is β, given a probability α of falsely claiming
its presence
Note 1 to entry: The term analytical sensitivity is sometimes used to mean detection limit, but such usage is now
discouraged. See ISO 18113-1:2009, A.2.7 and A.2.8 for further information.
Note 2 to entry: In a nucleic acid-based identification examination, the lowest concentration or content of the
target organism per defined amount of matrix that can be consistently detected under the experimental
conditions specified in the method.
Note 3 to entry: In molecular methods and quantitative molecular methods, the lowest concentration of
measurand that can be consistently detected (typically, in >95 % of samples (3.44) tested under routine clinical
laboratory conditions) and in a defined type of sample (3.44).
Note 4 to entry: This concentration must yield an assay value that can be reproducibly distinguished from values
obtained with samples (3.44) that do not contain the measurand.
[SOURCE: ISO 18113-1:2009, A.3.14, modified — new notes to entry added.]
3.26
limit of quantification
LOQ
lowest concentration or quantity of the nucleic acid target sequence (3.46) per defined volume that can
be measured with reasonable statistical certainty consistently under the experimental conditions
specified in the method
Note 1 to entry: Generally expressed in terms of the signal or measurement (true) value that will produce
estimates having a specified coefficient of variation (CV).
[SOURCE: ISO 16577:2016, 3.91, modified — replaced‚ content of the analyte of interest’ with ‘quantity
of the nucleic acid target sequence (3.46)’, ‘amount of matrix’ with ‘volume’ and ‘relative standard
deviation (RSD)’ with ‘coefficient of variation (CV).]
3.27
mastermix
mixture of reagents needed for nucleic acid amplification, except for the target DNA (3.17) and the
controls
[SOURCE: ISO 22174:2005, 3.4.18]
3.28
measurand
quantity intended to be measured
[SOURCE: VIM; JCGM 200; 2012]
EXAMPLE 1 Quantity of gene target measured by PCR (3.34) is influenced by the amplicon (3.2) size of the PCR
(3.34) assay and fragment size of the template (3.47) (∼
EXAMPLE 2 Denaturation (3.15) of DNA (3.17) in a sample (3.44) into ssDNA influences quantification by dPCR
as the two strands are partitioned separately.
Note 1 to entry: The specification of a measurant requires knowledge of the kind of quantity, including any
relevant component, and the chemical entities involved.
Note 2 to entry: In the second edition of the VIM and in IEC 60050-300:2001, the measurand is defined as the
‘particular quantity subject to measurement’.
6 © ISO 2020 – All rights reserved
---------------------- Page: 11 ----------------------
ISO 17822:2020(E)
Note 3 to entry: The measurement, including the measuring system and the conditions under which the
measurement is carried out, might change the phenomenon, body, or substance such that the quantity being
measured differs from the measurand as defined. In this, adequate correction is necessary.
[SOURCE: ISO/IEC guide 99:2007, 2.23, modified — Note 3 and examples have been modified, and Note 4
has been omitted.]
3.29
negative (PCR) control
reaction performed without target template (3.47)
[SOURCE: ISO 22174:2005, 3.5.6]
3.30
negative (process) control
target pathogen-free sample (3.44) of the collected specimen which is run through all stages of the
analytical process
Note 1 to entry: The nucleic-acid based examination process typically includes sample (3.44) preparation,
enrichment, nucleic acid (3.32) extraction and target amplification.
[SOURCE: ISO 22174:2005, 3.5.2, modified — Note 1 to entry modified.]
3.31
no template control
NTC
control reaction containing all reagents except the extracted test sample (3.44) template (3.47) nucleic
acid (3.32)
Note 1 to entry: This control is used to demonstrate the absence of contaminating nucleic acids [3.32]. Instead
of the template (3.47) DNA (3.17), for example, a corresponding volume of nucleic acid-free water is added to the
reaction. The term ‘PCR (3.34) reagent control’ is also sometimes used.
[SOURCE: ISO 20395:2019, 3.20]
3.32
nucleic acid
macromolecule that is the medium for genetic information or acts as an agent in expressing the
information
Note 1 to entry: There are two types of nucleic acid, DNA (3.17) and
...
INTERNATIONAL ISO
STANDARD 17822
First edition
2020-11
Corrected version
2020-12
In vitro diagnostic test systems —
Nucleic acid amplification-based
examination procedures for detection
and identification of microbial
pathogens — Laboratory quality
practice guide
Systèmes d'essai pour diagnostic in vitro — Modes opératoires
d'examen in vitro qualitatifs fondés sur l'acide nucléique pour la
détection et l'identification d'agents pathogènes microbiens — Guide
pratique sur la qualité dans les laboratoires
Reference number
ISO 17822:2020(E)
©
ISO 2020
---------------------- Page: 1 ----------------------
ISO 17822:2020(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
---------------------- Page: 2 ----------------------
ISO 17822:2020(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General laboratory requirement for microbial pathogens NAAT .10
4.1 General laboratory risk management and biosafety requirements .10
4.2 General laboratory set ups for pathogen detection .10
4.3 Commercial equipment (including software programs) .12
4.4 Laboratory personnel .12
5 Planning and implementation of pathogen NAAT assay .12
5.1 Quality control material .13
5.1.1 Examination of quality control material .13
5.1.2 Defining target sequence .15
5.2 Verification and validation .16
6 Verification or validation of test systems .16
6.1 Predicate assay selection by method comparison.16
7 Assay design and development of LDT .17
7.1 General .17
7.1.1 Definition of customer/patient’s and stakeholder needs of the intended
use of the assay .17
7.1.2 General criteria for Verification of assay .18
7.1.3 Specific criteria for verification of assay design input specifications .18
7.1.4 Validation of intended use .19
7.2 Diagnostic workflow analysis for Nucleic NAAT procedure .19
7.2.1 Pre-analytical workflow requirements .19
7.2.2 Analytical workflow requirements .20
7.2.3 Post-analytical workflow requirements .22
7.3 Verification and validation performance characteristics .23
7.3.1 Range of detection .23
7.3.2 Test accuracy (Trueness and Precision) .23
7.4 Analytical sensitivity / limit of detection .25
7.4.1 Validation of assay .26
8 Implementation and use in the laboratory .27
9 Reporting and interpretation of results .27
10 Quality assurance procedures .28
10.1 Performance monitoring and optimization of the assay .28
10.2 Inter-laboratory comparison .29
Annex A (informative) Pre-analytical consideration for sample preparation .30
Annex B (informative) Verification and validation of assays .37
Bibliography .38
© ISO 2020 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO 17822:2020(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 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 212, Clinical laboratory testing and in
vitro diagnostic test systems.
This first edition of ISO 17822 cancels and replaces ISO/TS 17822-1:2014, which has been technically
revised. The main changes are as follows:
— Clause 4 has been updated and merged from ISO/TS 17822-1:2014.
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
This corrected version of ISO 17822:2020 incorporates the following correction:
— The title on the cover page and page 1 has been corrected to remove the part name.
iv © ISO 2020 – All rights reserved
---------------------- Page: 4 ----------------------
ISO 17822:2020(E)
Introduction
Nucleic acid amplification-based tests (NAATs) are now commonly used in in vitro diagnostic (IVD)
tools in laboratory medicine for the detection, identification and quantification of microbial pathogens.
The NAAT result is influenced by all steps of the entire diagnostic workflow (i.e. pre-examination,
examination, post-examination). Therefore, this document considers all critical aspects of the entire
diagnostic workflow when designing, developing and implementing and using a specific microbial
pathogen NAAT examination.
NAAT examinations include PCR technology as well as other amplification-based technologies such
as, but not limited to, loop-mediated isothermal amplification (LAMP), transcription-mediated
amplification (TMA) and strand displacement amplification (SDA).
This document covers the implementation of commercially available IVD(s) into the medical laboratory
routine use as well as the design, development and implementation of laboratory developed tests (LDT).
This document will address the additional specific considerations, requirements and recommendations
for the detection of microbial pathogens with sampling, nucleic acid extraction, genetic heterogeneity
and the laboratory containment category which is required.
Due to high analytical sensitivity of nucleic acid-based examination procedures, special attention to
their design, development and use is required. This includes verification of analytical and validation of
clinical performance characteristics.
In this document, the following verbal forms are used:
— “shall” indicates a requirement;
— “should” indicates a recommendation;
— “may” indicates a permission;
— “can” indicates a possibility or a capability.
© ISO 2020 – All rights reserved v
---------------------- Page: 5 ----------------------
INTERNATIONAL STANDARD ISO 17822:2020(E)
In vitro diagnostic test systems — Nucleic acid
amplification-based examination procedures for detection
and identification of microbial pathogens — Laboratory
quality practice guide
1 Scope
This document describes the particular clinical laboratory practice requirements to ensure the quality
of detection, identification and quantification of microbial pathogens using nucleic acid amplification
tests (NAAT).
It is intended for use by laboratories that develop, and/or implement and use, or perform NAAT for
medical, research or health-related purposes. This document does not apply to the development of in
vitro diagnostic (IVD) medical devices by manufacturers. However, it does include verification and
validation of such devices and/or the corresponding processes when implemented and used by the
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 15189, Medical laboratories — Requirements for quality and competence
ISO 15190, Medical laboratories — Requirements for safety
3 Terms and definitions
For the purposes of this document, the following terms and definitions 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
accuracy
closeness of agreement between a test result or measurement result and the true value
Note 1 to entry: In practice, the accepted reference value is substituted for the true value.
Note 2 to entry: The term “accuracy”, when applied to a set of test or measurement results, involves a combination
of random components and a common systematic error or bias component.
Note 3 to entry: Accuracy refers to a combination of trueness and precision.
[SOURCE: ISO 3534-2:2006]
© ISO 2020 – All rights reserved 1
---------------------- Page: 6 ----------------------
ISO 17822:2020(E)
3.2
amplification product
amplicon
specific DNA (3.17) fragment produced by a DNA-amplification technology, such as the polymerase chain
reaction (PCR) (3.34)
[SOURCE: ISO 13495:2013, 3.3.1]
3.3
analytical specificity
specificity
capability of a measuring system, using a specified measurement procedure, to provide measurement
results for one or more measurands (3.28) which do not depend on each other nor on any other quantity
in the system undergoing measurement
[21]
Note 1 to entry: Lack of analytical specificity is called analytical interference (see ISO 18113-1:2009, A.3.2 ).
Note 2 to entry: Specificity of a measurement procedure should not be confused with clinical specificity (SOURCE:
[21]
ISO 18113-1:2009, A.3.16 ).
[22]
Note 3 to entry: VIM; JCGM 200; 2012 uses the term selectivity for this concept instead of specificity.
Note 4 to entry: For qualitative and semiquantitative examination procedures, analytical specificity is determined
by the ability to obtain negative results in concordance with negative results obtained by the reference method.
[SOURCE: ISO 18113-1:2009, A.3.4]
3.4
biorisk
probability or chance that a particular adverse event (in the context of this document: accidental
infection or unauthorized access, loss, theft, misuse, diversion or intentional release), possibly leading
to harm, will occur
[SOURCE: WHO Biorisk management , Laboratory biosecurity guidance , September 2006]
3.5
biosafety
describes the containment principles, technologies and practices that are implemented to prevent the
unintentional exposure to pathogens and toxins, or their accidental release
[SOURCE: WHO Biorisk management Laboratory biosecurity guidance September 2006]
3.6
biosecurity
set of preventive measures and actions to reduce the risk of intentional or unintentional transmission
of infectious diseases
Note 1 to entry: Biosecurity encompasses the prevention of the intentional removal (theft) of biological materials
from laboratories.
Note 2 to entry: These preventive measures are a combination of systems and practices implemented in
laboratories against the use of dangerous pathogens and toxins for malicious use to prevent the spread of these
biological agents.
2 © ISO 2020 – All rights reserved
---------------------- Page: 7 ----------------------
ISO 17822:2020(E)
3.7
calibration
operation that, under specified conditions, in a first step, established a relation between the quantity
values with measurement uncertainties provided by measurement standards and corresponding
indications with associated measurement uncertainties and, in second step, uses this information to
establish a relation for obtaining a measurement result from an indication
Note 1 to entry: according to US Code of Federal Regulations, calibration is a process of testing and adjusting an
instrument or test system to establish a correlation between the measurement response and the concentration
[20]
or amount of the substance that is being measured by the test procedure (modified from 42CFR 493.1218) .
[SOURCE: VIM; JCGM 200; 2012]
3.8
certified reference material
CRM
reference material (RM) (3.41) characterized by a metrologically valid procedure for one or more
specified properties, accompanied by a RM (3.41) certificate that provides the value of the specified
property, its associated uncertainty, and a statement of metrological traceability
Note 1 to entry: The concept of value includes a nominal property or a qualitative attribute such as identity or
sequence. Uncertainties for such attributes may be expressed as probabilities or levels of confidence.
Note 2 to entry: Metrologically valid procedures for the production and certification of RMs (3.41) are given in,
[17]
among others, ISO 17034 .
[19]
Note 3 to entry: ISO/IEC Guide 99:2007 has an analogous definition.
[SOURCE: ISO Guide 30, 2.1.2]
3.9
clinical performance
ability of an in vitro diagnostic examination procedure to yield results that
are correlated with a specific clinical condition or physiological state in accordance with the target
population and intended user
Note 1 to entry: Although sometimes referred to as diagnostic performance or clinical validity; clinical
performance is the harmonized term endorsed by the Global Harmonization Task Force (GHTF) and its successor,
the International Medical Devices Regulators Forum (IMDRF).
Note 2 to entry: Evaluation of clinical performance often relies on the outcome of other types of clinical
examinations to define "true positive or true negative" results.
[SOURCE: GHTF/ SG5/N 6:2012, 4.4.2, modified — medical device has been changed to — examination
procedure and particularly has been changed to — specific.]
3.10
clinical sensitivity
diagnostic sensitivity
ability of an in vitro diagnostic examination procedure to identify the presence
of a target marker associated with a specific disease or condition
Note 1 to entry: Also defined as percent positivity in samples (3.44) where the target marker is known to be
present.
Note 2 to entry: Diagnostic sensitivity is expressed as a percentage (number fraction multiplied by 100),
calculated as 100 × the number of true positive values (TP) divided by the sum of the number of true positive
values (TP) plus the number of false negative values (FP), or 100 × TP/(TP + FN). This calculation is based on a
study design where only one sample (3.44) is taken from each subject.
Note 3 to entry: The target condition is defined by criteria independent of the examination procedure under
consideration.
© ISO 2020 – All rights reserved 3
---------------------- Page: 8 ----------------------
ISO 17822:2020(E)
[SOURCE: ISO 18113-1:2009, A.3.15]
3.11
clinical specificity
diagnostic specificity
ability of an in vitro diagnostic examination procedure to recognise the absence
of a target marker associated with a specific disease or condition
Note 1 to entry: Also defined as percent negativity in samples (3.44) where the target marker is known to be absent.
Note 2 to entry: Clinical specificity is expressed as a percentage (number fraction multiplied by 100), calculated
as 100× the number of true negative values (TN) divided by the sum of the number of true negative plus the
number of false positive (FP) values, or 100 × TN/(TN + FP). This calculation is based on a study design where
only one sample (3.44) is taken from each subject.
Note 3 to entry: The target condition is defined by criteria independent of the examination procedure under
consideration.
[SOURCE: ISO 18113-1:2009, A.3.16]
3.12
complementary DNA
cDNA
single-stranded DNA (3.17) that is complementary to a given RNA (3.42) and synthesized in the presence
of reverse transcriptase to serve as a template (3.47) for synthesis of DNA (3.17) copies
3.13
contamination
introduction of an undesirable substance or matter
3.14
cut-off value
quantity value used as a limit to identify samples (3.44) that indicate the presence or the absence of a
specific disease, condition or measurand (3.28)
Note 1 to entry: Defines which measurement results are reported as positive and which are reported as negative.
Note 2 to entry: Measurement results near the cut-off value can be considered inconclusive.
Note 3 to entry: The selection of the cut-off value determines the clinical specificity (3.11) and clinical sensitivity
(3.10) of the examination.
3.15
denaturation
physical and/or chemical treatment which results in the separation of nucleic acid double helices
Note 1 to entry: denaturation of DNA (3.17) results in separation of double-stranded DNA (3.17) into single-
stranded DNA (3.17).
[SOURCE: ISO 21572:2013, 3.1.6 — modified, term ”denaturation of proteins” has been changed to
”denaturation”, and ” the POI or” has been deleted. Note 1 to entry has been added.]
3.16
deoxyribonucleoside triphosphate
dNTP
solution containing dATP, dCTP, dGTP, dTTP and/or dUTP
[SOURCE: ISO 22174:2005, 3.3.7]
4 © ISO 2020 – All rights reserved
---------------------- Page: 9 ----------------------
ISO 17822:2020(E)
3.17
DNA
deoxyribonucleic acid
polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded
(ssDNA) form
[SOURCE: ISO 22174:2005, 3.1.2]
3.18
DNA polymerase for PCR
thermostable enzyme which catalyses repeated DNA (3.17) synthesis
[SOURCE: ISO 22174:2005, 3.4.17]
3.19
DNA sequencing
determining the order of nucleotide bases (adenine, guanine, cytosine, and thymine) in a molecule of
DNA (3.17)
Note 1 to entry: Sequence is generally described from the 5’ end.
3.20
hybridization
specific binding of complementary nucleic acid (3.32) sequences under suitable reaction conditions
[SOURCE: ISO 22174:2005, 3.6.3]
3.21
inhibition
reduction of amplification or interference with detection process that can lead to false negative results
or reduced quantity
3.22
interfering substances
endogenous or exogenous substances in clinical specimens/samples (3.44) that can alter
an examination result
[SOURCE: ISO 20186:2019-1, 3.15 modified]
3.23
inhouse assay
laboratory developed test
LDT
type of in vitro diagnostic test that is designed, manufactured and used within a single laboratory
Note 1 to entry: Inhouse assay/LDT needs to be validated for its intended use before putting into service.
3.24
linearity
ability of a method of analysis, within a certain range, to provide an instrumental response or results
proportional to the quantity of nucleic acid target sequence (3.46) to be determinded in the laboratory
sample (3.44)
Note 1 to entry: In the case of qPCR, the quantification cycle (also termed cycle threshold or crossing point) is
inversely proportional to the quantity of nucleic acid target sequence. (3.46).
Note 2 to entry: The term linearity is frequently linked with the linear range of the method and refers to the
ability of a method to give a response or result that is directly propotional to the concentration of the nucleic acid
target sequence (3.46).
[SOURCE: ISO 16577:2016, 3.92 modified — Notes 1 and 2 to entry added; ‘quantity of analyte’ replaced
with ‘quantity of the nucleic acid target sequence’ (3.46).]
© ISO 2020 – All rights reserved 5
---------------------- Page: 10 ----------------------
ISO 17822:2020(E)
3.25
limit of detection
LOD
measured quantity value, obtained by a given measurement procedure, for which the probability of
falsely claiming the absence of a component in a material is β, given a probability α of falsely claiming
its presence
Note 1 to entry: The term analytical sensitivity is sometimes used to mean detection limit, but such usage is now
discouraged. See ISO 18113-1:2009, A.2.7 and A.2.8 for further information.
Note 2 to entry: In a nucleic acid-based identification examination, the lowest concentration or content of the
target organism per defined amount of matrix that can be consistently detected under the experimental
conditions specified in the method.
Note 3 to entry: In molecular methods and quantitative molecular methods, the lowest concentration of
measurand that can be consistently detected (typically, in >95 % of samples (3.44) tested under routine clinical
laboratory conditions) and in a defined type of sample (3.44).
Note 4 to entry: This concentration must yield an assay value that can be reproducibly distinguished from values
obtained with samples (3.44) that do not contain the measurand.
[SOURCE: ISO 18113-1:2009, A.3.14, modified — new notes to entry added.]
3.26
limit of quantification
LOQ
lowest concentration or quantity of the nucleic acid target sequence (3.46) per defined volume that can
be measured with reasonable statistical certainty consistently under the experimental conditions
specified in the method
Note 1 to entry: Generally expressed in terms of the signal or measurement (true) value that will produce
estimates having a specified coefficient of variation (CV).
[SOURCE: ISO 16577:2016, 3.91, modified — replaced‚ content of the analyte of interest’ with ‘quantity
of the nucleic acid target sequence (3.46)’, ‘amount of matrix’ with ‘volume’ and ‘relative standard
deviation (RSD)’ with ‘coefficient of variation (CV).]
3.27
mastermix
mixture of reagents needed for nucleic acid amplification, except for the target DNA (3.17) and the
controls
[SOURCE: ISO 22174:2005, 3.4.18]
3.28
measurand
quantity intended to be measured
[SOURCE: VIM; JCGM 200; 2012]
EXAMPLE 1 Quantity of gene target measured by PCR (3.34) is influenced by the amplicon (3.2) size of the PCR
(3.34) assay and fragment size of the template (3.47) (∼
EXAMPLE 2 Denaturation (3.15) of DNA (3.17) in a sample (3.44) into ssDNA influences quantification by dPCR
as the two strands are partitioned separately.
Note 1 to entry: The specification of a measurant requires knowledge of the kind of quantity, including any
relevant component, and the chemical entities involved.
Note 2 to entry: In the second edition of the VIM and in IEC 60050-300:2001, the measurand is defined as the
‘particular quantity subject to measurement’.
6 © ISO 2020 – All rights reserved
---------------------- Page: 11 ----------------------
ISO 17822:2020(E)
Note 3 to entry: The measurement, including the measuring system and the conditions under which the
measurement is carried out, might change the phenomenon, body, or substance such that the quantity being
measured differs from the measurand as defined. In this, adequate correction is necessary.
[SOURCE: ISO/IEC guide 99:2007, 2.23, modified — Note 3 and examples have been modified, and Note 4
has been omitted.]
3.29
negative (PCR) control
reaction performed without target template (3.47)
[SOURCE: ISO 22174:2005, 3.5.6]
3.30
negative (process) control
target pathogen-free sample (3.44) of the collected specimen which is run through all stages of the
analytical process
Note 1 to entry: The nucleic-acid based examination process typically includes sample (3.44) preparation,
enrichment, nucleic acid (3.32) extraction and target amplification.
[SOURCE: ISO 22174:2005, 3.5.2, modified — Note 1 to entry modified.]
3.31
no template control
NTC
control reaction containing all reagents except the extracted test sample (3.44) template (3.47) nucleic
acid (3.32)
Note 1 to entry: This control is used to demonstrate the absence of contaminating nucleic acids [3.32]. Instead
of the template (3.47) DNA (3.17), for example, a corresponding volume of nucleic acid-free water is added to the
reaction. The term ‘PCR (3.34) reagent control’ is also sometimes used.
[SOURCE: ISO 20395:2019, 3.20]
3.32
nucleic acid
macromolecule that is the medium for genetic information or acts as an agent in expressing the
information
Note 1 to entry: Th
...
DRAFT INTERNATIONAL STANDARD
ISO/DIS 17822-2
ISO/TC 212 Secretariat: ANSI
Voting begins on: Voting terminates on:
Corrected version
2019-11-08 2020-01-31
2019-11
In vitro diagnostic test systems — Nucleic acid
amplification- based examination procedures for detection
and identification of microbial pathogens —
Part 2:
Laboratory quality practice guide
ICS: 11.100.01
THIS DOCUMENT IS A DRAFT CIRCULATED
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
BEING ACCEPTABLE FOR INDUSTRIAL,
This document is circulated as received from the committee secretariat.
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 17822-2:2019(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 2019
---------------------- Page: 1 ----------------------
ISO/DIS 17822-2:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019
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
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/DIS 17822-2:2019(E)
Contents
Foreword . 4
Introduction. 5
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General laboratory requirement for microbial pathogens NAAT . 11
4.1 General laboratory risk management and biosafety requirements . 11
4.2 General laboratory set ups for pathogen detection . 11
4.3 Commercial equipment (including software programs) . 13
4.4 Laboratory personnel . 14
5 Planning and implementation of pathogen NAAT assay . 14
5.1 Quality control material . 14
5.2 Verification and validation . 17
6 Verification or validation of test systems . 18
6.1 Predicate assay selection by method comparison . 18
7 Assay design and development of LDT . 18
7.1 General . 18
7.2 Diagnostic workflow analysis for Nucleic NAAT procedure . 21
7.3 Verification and validation performance characteristics . 24
7.4 Analytical sensitivity / limit of detection . 27
8 Implementation and use in the laboratory . 29
9 Reporting and interpretation of results . 29
10 Quality assurance procedures . 30
10.1 Performance monitoring and optimization of the assay . 30
10.2 Inter-laboratory comparison . 30
Annex A (informative) Pre-analytical requirements for sample preparation . 32
Annex B (informative) Verification and validation of assays . 39
Bibliography . 40
© ISO 2019 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO/DIS 17822-2:2019(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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International
Standards adopted by the technical committees are circulated to the member bodies for voting.
Publication as an International Standard requires approval by at least 75 % of the member bodies
casting a vote.
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.
ISO 17822**-2 was prepared by Technical Committee ISO/TC 212, Clinical laboratory testing and in
vitro diagnostic test systems, Working Group 4
iv © ISO 2019 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/DIS 17822-2:2019(E)
Introduction
Nucleic acid amplification-based tests (NAATs) are now commonly used in in vitro diagnostic (IVD)
tools in laboratory medicine for the detection, identification and quantification of microbial pathogens.
The NAAT result is influenced by all steps of the entire diagnostic workflow (i.e. pre-examination,
examination, post-examination). Therefore, this document considers all critical aspects of the entire
diagnostic workflow when designing, developing and implementing and using a specific microbial
pathogen NAAT examination.
NAAT examinations include PCR technology as well as other amplification-based technologies such as,
but not limited to, loop-mediated isothermal amplification (LAMP), transcription-mediated
amplification (TMA) and strand displacement amplification (SDA).
This document covers the implementation of commercially available IVD(s) into the medical laboratory
routine use as well as the design, development and implementation of laboratory developed tests
(LDT).
This will address the additional considerations associated with the detection of microbial pathogens
such as challenges associated with sampling, nucleic acid extraction, genetic heterogeneity and the
laboratory containment category which is required.
Due to high analytical sensitivity of nucleic acid-based examination procedures, special attention to
their design, development and use is required. This includes verification of analytical and validation of
clinical performance characteristics.
In this document, the following verbal forms are used:
– “shall” indicates a requirement;
– “should” indicates a recommendation;
– “may” indicates a permission;
– “can” indicates a possibility or a capability.
© ISO 2019 – All rights reserved v
---------------------- Page: 5 ----------------------
ISO/DIS 17822-2:2019(E)
In vitro diagnostic test systems — Nucleic acid amplification-based
examination procedures for detection and identification of microbial
pathogens — Part 2: Laboratory quality practice guide
1 Scope
This document describes the particular clinical laboratory practice requirements to ensure the quality
of detection, identification and quantification of microbial pathogens using nucleic acid amplification
tests (NAAT).
It is intended for use by laboratories that develop, and/or implement and use, or perform NAAT for
medical, research or health-related purposes. This document does not apply to the development of in
vitro diagnostic (IVD) medical devices by manufacturers. However, it does include verification and
validation of such devices and/or the corresponding processes when implemented and used by the
laboratories. In vitro diagnostic test systems — Nucleic acid amplification-based examination
procedures for detection and identification of microbial pathogens — Part 2: Laboratory quality
practice guide.
2 Normative references
The following documents, in whole or in part, are normatively referenced in 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 15189, Medical laboratories — Requirements for quality and competence
ISO 15190, Medical laboratories — Requirements for safety
ISO 18113-2, In vitro diagnostic medical devices — Information supplied by the manufacturer (labelling)
— Part 2: In vitro diagnostic reagents for professional use
ISO 35001 Laboratory Biorisk Management, standard under development
3 Terms and definitions
For the purposes of this document, the following terms and definitions 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 https://www.iso.org/obp
3.01
accuracy
closeness of agreement between a test result or measurement result and the true value
Note 1 to entry: In practice, the accepted reference value is substituted for the true value.
Note 2 to entry: The term “accuracy”, when applied to a set of test or measurement results, involves a combination
of random components and a common systematic error or bias component.
Note 3 to entry: Accuracy refers to a combination of trueness and precision.
[SOURCE: ISO 3534-2:2006, 32]
© ISO 2019 – All rights reserved 1
---------------------- Page: 6 ----------------------
ISO/DIS 17822-2:2019(E)
3.02
amplification product
amplicon
specific DNA [3.17] fragment produced by a DNA-amplification technology, such as the polymerase chain
reaction (PCR) [3.34]
[SOURCE: ISO 13495:2013, 3.3.1]
3.03
analytical specificity
specificity
capability of a measuring system, using a specified measurement procedure, to provide measurement
results for one or more measurands [3.28] which do not depend on each other nor on any other quantity
in the system undergoing measurement
NOTE 1 to entry: Lack of analytical specificity is called analytical interference (see ISO 18113-1 A.3.2 [21]).
NOTE 2 to entry: Lack of analytical specificity in immunochemistry measurement procedures can be due to cross-
reactivity (see ISO 18113-1 A.3.12[21]).).
NOTE 3 to entry: Specificity of a measurement procedure should not be confused with clinical specificity
(SOURCE: ISO 18113-1 A.3.16 [21]).
NOTE 4 to entry: VIM; JCGM 200; 2012 [22] uses the term selectivity for this concept instead of specificity.
NOTE 5 to entry: For qualitative and semiquantitative examination procedures, analytical specificity is
determined by the ability to obtain negative results in concordance with negative results obtained by the
reference method.
[SOURCE: ISO 18113-1:2009, A.3.4 21]
3.04
biorisk
probability or chance that a particular adverse event (in the context of this document: accidental
infection or unauthorized access, loss, theft, misuse, diversion or intentional release), possibly leading
to harm, will occur.
SOURCE: WHO Biorisk management , Laboratory biosecurity guidance , September 2006, 11
3.05
biosafety
describes the containment principles, technologies and practices that are implemented to prevent the
unintentional exposure to pathogens and toxins, or their accidental release.
[SOURCE: WHO Biorisk management Laboratory biosecurity guidance September 2006, definitions[11]]
3.06
biosecurity
set of preventive measures and actions to reduce the risk of intentional or unintentional transmission of
infectious diseases. Biosecurity encompasses the prevention of the intentional removal (theft) of
biological materials from laboratories. These preventive measures are a combination of systems and
practices implemented in laboratories against the use of dangerous pathogens and toxins for malicious
use to prevent the spread of these biological agents
2 © ISO 2019 – All rights reserved
---------------------- Page: 7 ----------------------
ISO/DIS 17822-2:2019(E)
Note 1 to entry: Biosecurity encompasses the prevention of the intentional removal (theft) of biological materials
from laboratories
Note 2 to entry: These preventive measures are a combination of systems and practices implemented in
laboratories against the use of dangerous pathogens and toxins for malicious use to prevent the spread of these
biological agents
SOURCE: http://www.sciencemag.org/cgi/content/full/295/5552/44a Meyerson and Reaser 2002,
Science 295: 44, modified
3.07
calibration
standard
operation that, under specified conditions, in a first step, established a relation between the quantity
values with measurement uncertainties provided by measurement standards and corresponding
indications with associated measurement uncertainties and, in second step, uses this information to
establish a relation for obtaining a measurement result from an indication
NOTE 1 to entry: according to US Code of Federal Regulations, calibration is a process of testing and adjusting an
instrument or test system to establish a correlation between the measurement response and the concentration or
amount of the substance that is being measured by the test procedure (modified from 42CFR 493.1218) [20]
[SOURCE: VIM; JCGM 200; 2012 [22]]
3.08
certified reference material
CRM
reference material (RM) [3.41] characterized by a metrologically valid procedure for one or more
specified properties, accompanied by a RM [3.41] certificate that provides the value of the specified
property, its associated uncertainty, and a statement of metrological traceability
Note 1 to entry: The concept of value includes a nominal property or a qualitative attribute such as identity or
sequence. Uncertainties for such attributes may be expressed as probabilities or levels of confidence
Note 2 to entry: Metrologically valid procedures for the production and certification of RMs [3.41] are given in,
among others, ISO 17034 [17].
Note 3 to entry: ISO/IEC Guide 99:2007[19] has an analogous definition (5.14).
[Source: ISO guide 30, 2.1.2 [18]]
3.09
clinical performance
(laboratory medicine) ability of an in vitro diagnostic examination procedure to yield results that are
correlated with a specific clinical condition or physiological state in accordance with the target
population and intended user
NOTE 1 to entry: Although sometimes referred to as diagnostic performance or clinical validity; clinical
performance is the harmonized term endorsed by the Global Harmonization Task Force (GHTF) and its successor,
the International Medical Devices Regulators Forum (IMDRF).
NOTE 2 to entry: Evaluation of clinical performance often relies on the outcome of other types of clinical
examinations to define "true positive or true negative" results.
[SOURCE: GHTF/ SG5/N 6:2012[23], 4.4.2, modified – medical device has been changed
to―examination procedure and particularly has been changed to ―specific.].
© ISO 2019 – All rights reserved 3
---------------------- Page: 8 ----------------------
ISO/DIS 17822-2:2019(E)
3.10
clinical sensitivity
diagnostic sensitivity
(laboratory medicine) ability of an in vitro diagnostic examination procedure to identify the presence of
a target marker associated with a specific disease or condition
NOTE 1 to entry: Also defined as percent positivity in samples [3.44] where the target marker is known to be
present.
NOTE 2 to entry: Diagnostic sensitivity is expressed as a percentage (number fraction multiplied by 100),
calculated as 100 × the number of true positive values (TP) divided by the sum of the number of true positive
values (TP) plus the number of false negative values (FP), or 100 × TP/(TP + FN). This calculation is based on a
study design where only one sample [3.44] is taken from each subject.
NOTE 3 to entry: The target condition is defined by criteria independent of the examination procedure under
consideration.
[SOURCE: ISO 18113-1:2009, A.3.15 [21]]
3.11
clinical specificity
diagnostic specificity
(laboratory medicine) ability of an in vitro diagnostic examination procedure to recognise the absence
of a target marker associated with a specific disease or condition
NOTE 1 to entry: Also defined as percent negativity in samples [3.44] where the target marker is known to be
absent.
NOTE 2 to entry: Clinical specificity is expressed as a percentage (number fraction multiplied by 100), calculated
as 100 × the number of true negative values (TN) divided by the sum of the number of true negative plus the
number of false positive (FP) values, or 100 × TN/(TN + FP). This calculation is based on a study design where
only one sample [3.44] is taken from each subject.
NOTE 3 to entry: The target condition is defined by criteria independent of the examination procedure under
consideration.
[SOURCE: ISO 18113-1:2009, A.3.16, [21]]
3.12
complementary DNA
cDNA
single-stranded DNA [3.17] that is complementary to a given RNA [3.42] and synthesized in the
presence of reverse transcriptase to serve as a template [3.47] for synthesis of DNA [3.17] copies.
3.13
contamination
introduction of an undesirable substance or matter
3.14
cut-off value
quantity value used as a limit to identify samples [3.44] that indicate the presence or the absence of a
specific disease, condition or measurand [3.28]
NOTE 1 to entry: Defines which measurement results are reported as positive and which are reported as negative.
NOTE 2 to entry: Measurement results near the cut-off value can be considered inconclusive.
4 © ISO 2019 – All rights reserved
---------------------- Page: 9 ----------------------
ISO/DIS 17822-2:2019(E)
NOTE 3 to entry: The selection of the cut-off value determines the clinical specificity [3.11] and clinical sensitivity
[3.10] of the examination.
3.15
denaturation
physical and/or (bio) chemical treatment which destroys or modifies the structural, functional,
enzymatic, or antigenic properties of the analyte
NOTE 1 to entry: denaturation of DNA [3.17]results in separation of double-stranded DNA [3.17] into single-
stranded DNA [3.17].
[SOURCE: ISO 21572:2013, definition 3.1.6, modified, term ”denaturation of proteins” has been changed
to ”denaturation”, and ” the POI or” has been deleted. Note 1 to entry has been added. [24]]
3.16
deoxyribonucleoside triphosphate
dNTP
solution containing dATP, dCTP, dGTP, dTTP and/or dUTP
[SOURCE: ISO 22174:2005, 3.3.7[20]]
3.17
DNA
deoxyribonucleic acid
polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded (ssDNA)
form
[SOURCE: ISO 22174:2005, 3.1.2 [20]]
3.18
DNA polymerase for PCR
thermostable enzyme which catalyses repeated DNA [3.17] synthesis
[SOURCE: ISO 22174:2005, 3.4.17 [20]]
3.19
DNA sequencing
determining the order of nucleotide bases (adenine, guanine, cytosine, and thymine) in a molecule of
DNA [3.17]
NOTE 1 to entry: Sequence is generally described from the 5’ end.
3.20
hybridization
specific binding of complementary nucleic acid [3.32] sequences under suitable reaction conditions
[SOURCE: ISO 22174:2005, 3.6.3 [20]]
3.21
inhibition
reduction of amplification or interference with detection process that can lead to false negative results
or reduced quantity.
© ISO 2019 – All rights reserved 5
---------------------- Page: 10 ----------------------
ISO/DIS 17822-2:2019(E)
3.22
interfering substances
endogenous or exogenous substances in clinical specimens/samples [3.44] that can alter
an examination result.
SOURCE: ISO 20186:2019-1 [36], 3.15 modified)
3.23
inhouse assay
laboratory developed test
LDT
type of in vitro diagnostic test that is designed, manufactured and used within a single laboratory
Note 1 to entry: Inhouse assay/LDT needs to be validated for its intended use before putting into service.
3.24
linearity
ability of a method of analysis, within a certain range, to provide an instrumental response or results
proportional to the quantity of nucleic acid target sequence [3.46] to be determinded in the laboratory
sample [3.44]
Note 1 to entry: In the case of qPCR, the cycle threshold is proportional to the logarithm to the base 10 of the
quantity of nucleic acid target sequence [3.46].
Note 2 to entry: The term linearity is frequently linked with the linear range of the method and refers to the ability
of a method to give a response or result that is directly propotional to the concentration of the nucleic acid target
sequence [3.46].
SOURCE: ISO 16577:2016, 3.92 modified – Notes 1 and 2 to entry added; ‘quantity of analyte’ replaced
with ‘quantity of the nucleic acid target sequence’ [3.46] [36].
3.25
limit of detection
LOD
measured quantity value, obtained by a given measurement procedure, for which the probability of
falsely claiming the absence of a component in a material is β, given a probability α of falsely claiming
its presence
NOTE 1 to entry: The term analytical sensitivity is sometimes used to mean detection limit, but such usage is now
discouraged. See ISO 18113-1, A.2.7 and A.2.8 for further information.
NOTE 2 to entry: In a nucleic acid-based identification examination, the lowest concentration or content of the
target organism per defined amount of matrix that can be consistently detected under the experimental conditions
specified in the method.
[SOURCE: JCGM 200:2008, definition 4.18 (notes deleted) [22]]
[SOURCE: ISO 22174:2005, 3.1.8, [20]]
Note 3 to entry: in molecular methods and quantitative molecular methods, the lowest concentration of
measurand that can be consistently detected (typically, in >95% of samples [3.44] tested under routine clinical
laboratory conditions) and in a defined type of sample [3.44].
NOTE 4 to entry: This concentration must yield an assay value that can be reproducibly distinguished from values
obtained with samples [3.44] that do not contain the measurand.
6 © ISO 2019 – All rights reserved
---------------------- Page: 11 ----------------------
ISO/DIS 17822-2:2019(E)
3.26
limit of quantification
LOQ
lowest concentration or quantity of the nucleic acid target sequence [3.46] per defined volume that can
be measured with reasonable statistical certainty consistently under the experimental conditions
specified in the method
Note 1 to entry: Generally expressed in terms of the signal or measurement (true) value that will produce
estimates having a specified coefficient of variation (CV).
SOURCE: ISO 16577:2016, 3.91, modified – replaced ‚content oft he analyte of interest’ with ‘quantity of
the nucleic acid target sequence [3.46]’, ‘amount of matrix’ with ‘volume’ and ‘relative standard
deviation (RSD)’ with ‘coefficient of variation (CV). [36]
3.27
mastermix
mixture of reagents needed for PCR [3.34], except for the target DNA [3.17] and the controls
[ISO 22174:2005, 3.4.18]
3.28
measurand
quantity intended to be measured (SOURCE: VIM; JCGM 200; 2012 [22])
NOTE 1 to entry: The specification of a measurant requires knowledge of the kind of quantity, including any
relevant component, and the chemical entities involved.
Note 2 to entry: In the second edition of the VIM and in IEC 60050-300:2001, the measurand is defined as the
‘particular quantity subject to measurement’.
Note 3 to entry: The measurement, including the measuring system and the conditions under which the
measurement is carried out, might change the phenomenon, body, or substance such that the quantity being
measured differs from the measurand as defined. In this, adequate correction is necessary.
EXAMPLE 1 Quantity of gene target measured by PCR [3.34] is influenced by the amplicon [3.02] size of the
PCR [3.34] assay and fragment size of the template [3.47] (
EXAMPLE 2 Denaturation [3.15] of DNA [3.17] in a sample [3.44] into ssDNA influences quantification by
dPCR as the two strands are partitioned separately.
SOURCE: ISO/IEC guide 99:2007, 2.23, modified – Note 3 and examples have been modified, and Note 4
has been omitted.
3.29
negative (PCR) control
reaction performed with nucleic acid-free water without any PCR [3.34] inhibitors
[SOURCE: ISO 22174:2005, 3.5.6 20]
3.30
negative (process) control
target pathogen-free sample [3.44] of the collected specimen which is run through all stages of the
analytical process
[SOURCE: ISO 22174:2005, 3.5.2 (modified) 20]
© ISO 2019 – All rights reserved 7
---------------------- Page: 12 ----------------------
ISO/DIS 17822-2:2019(E)
NOTE 1 to entry: The nucleic-acid based examination process typically includes sample [3.44] preparation,
enrichment, nucleic acid [3.32] extraction and target amplification.
3.31
no template control
NTC
control reaction containing all reagents except the extracted test sample [3.44] template [3.47] nucleic
acid [3.32]
NOTE 1 to entry: This control is used to demonstrate the absence of contaminating nucleic acids [3.32]. Instead of
the template [3.47] DNA [3.17], for example, a corresponding volume of nucleic acid-free water is added to the
reaction. The term ‘PCR [3.34] reagent control’ is also sometimes used.
[SOURCE: ISO 20395:2019, 3.20 12]
3.32
nucleic acid
macromolecule that is the medium for genetic information or acts as an agent in expressing the
information
NOTE 1 to entry: There are two types of nucleic acid, DNA [3.17] and RNA [3.42]
[SOURCE: ISO 22174:2005, 3.1.1 20]
3.33
nucleic acid extraction
separation of nucleic acid [3.32] from biological materials
NOTE 1 to entry: Generally to perform amplification and analysis of the nucleic acid [3.32].
3.34
polymerase chain reaction
PCR
enzymatic procedure which allows in vitro amplification of DNA [3.17]
[SOURCE: ISO 22174:2005, 3.4.1 20]
3.35
polynomial regression
A least squared regression using polynomials of various orders
Y=a+b1X (first-order polynomial or linear fit)
Y=a+ b1X+b2X2 (second- order polynomial), and
Y=a+ b1X+b2X2+b3X3 (third- order polynomial)
3.36
PCR-quality DNA
DNA [3.17] template [3.47] of sufficient length, purity, and quantity for performing PCR [3.34]
[SOURCE: ISO 24276:2006, definition 3.2.3, 28]
3.37
regulatory body approved assay
tests that are designed and developed by manufacturers and approved by regulatory body for
diagnostic purposes.
8 © ISO 2019 – All rights reserved
---------------------- Page: 13 ----------------------
ISO/DIS 17822-2:2019(E)
EXAMPLE: CE-labeled tests
NOTE 1 to entry: The CE marking is the manufacturer's declaration that the product meets the requirements of the
applicable EC directives.
3.38
reverse transcription
RT
process of making DNA [3.17] from an RNA [3.42] template [3.47], us
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.