ISO 17822:2020

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 2020
© ISO 2020
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ii © ISO 2020 – All rights reserved

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
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

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.
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]
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

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.
[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

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).]
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

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 RNA (3.42).
[SOURCE: ISO 22174:2005, 3.1.1]
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]
3.35
polynomial regression
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, 3.2.3]
3.37
regulatory body approved assay
tests that are designed and developed by manufacturers and approved by regulatory body for diagnostic
purposes.
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), using the enzymatic activity of a
reverse transcriptase associated with one or more oligonucleotide primers under a suitable set of
conditions
[SOURCE: ISO 16577:2016, 3.180]
3.39
modified IVD labeled assays
modified IVD labeled tests
tests that are designed and developed by manufacturers and approved by regulatory body or they meet
the requirements of the applicable EC directives for diagnostic purposes but in use of the laboratories
they have been changed
Note 1 to entry: Depending on the grade of change made to the original assay this assay needs to be validated again.
3.40
real time PCR
method, which combines PCR (3.34) and fluorescent probe detection of amplified product in the same
reaction vessel
3.41
reference material
RM
material, sufficiently homogeneous and stable with respect to one or more specified properties, which
has been established to be fit for its intended use in a measurement process
Note 1 to entry: RM is a generic term.
Note 2 to entry: Properties can be quantitative or qualitative, e.g. identity of substances or species.
Note 3 to entry: Uses may include the calibration (3.7) of a measurement system, assessment of a measurement
procedure, assigning values to other materials, and quality control.
[19]
Note 4 to entry: ISO/IEC Guide 99:2007 has an analogous definition (5.13), but restricts the term
”measurement” to apply to quantitative values. However, Note 3 of ISO/IEC Guide 99:2007, 5.13 (VIM; JCGM 200;
[22]
2012 ), specifically includes qualitative properties, called ”nominal properties”.
[SOURCE: ISO guide 30]
8 © ISO 2020 – All rights reserved

3.42
RNA
ribonucleic acid
polymer of ribonucleotides occurring in a double-stranded or single-stranded form
[SOURCE: ISO 22174:2005, 3.1.3]
3.43
robustness
ability of an assay to proceed optimally, despite slight variation in conditions
Note 1 to entry: to entry: Usually refers to PCR (3.34) in which amplification occurs despite slight changes in
reaction conditions, such as DNA (3.17) concentration.
3.44
sample
small portion or quantity, taken from a population or lot that is ideally a representative selection of
the whole
Note 1 to entry: Usually refers to PCR (3.34) in which amplification occurs despite slight changes in reaction
conditions, such as DNA (3.17) concentration.
[SOURCE: ISO 16577:2016, 3.185]
3.45
sequence database
biological database consisting of nucleic acid (3.32) sequences, protein sequences, or
other polymer sequences and associated annotation
Note 1 to entry: The annotation can relate to organism, species, function, mutations linked to particular diseases,
functional or structural features, bibliographic references, etc.
Note 2 to entry: Published genome sequences can be publically available, as it is a requirement of every scientific
journal that any published DNA (3.17) or RNA (3.42) or protein sequence must be deposited in a public database.
3.46
target sequence
nucleic acid target sequence
specific DNA (3.17) sequence targeted for detection, e.g. by PCR (3.34)
[SOURCE: ISO 16577:2016, 3.203]
3.47
template
strand of DNA (3.17) or RNA (3.42) that specifies the base sequence of a newly synthesized strand of
DNA (3.17) or RNA (3.42), the two strands being complementary
[SOURCE: ISO 16577:2016, 3.206]
3.48
unidirectional work flow
forward work flow
principle of material/sample (3.44) handling applied to ensure that the
laboratory sample (3.44), raw and processed test portion including amplified DNA (3.17) remain
physically segregated during the entire procedure
[SOURCE: ISO 24276:2006, 3.3.5 modified — “laboratory sample (3.44), raw and processed test portion”
has been changed to “the primary sample (3.44) and the processed sample (3.44)”, and “the whole
procedure” has been changed to “the examination procedure”]
3.49
validation
confirmation, through the provision of objective evidence, that the requirements for a specific intended
use or application have been fulfilled
Note 1 to entry: The term "validated" is used to designate the corresponding status.
[31]
Note 2 to entry: Adapted from ISO 9000:2015 .
Note 3 to entry: See Annex B for additional information.
[SOURCE: ISO 15189:2012, 3.26]
3.50
verification
confirmation, through provision of objective evidence, that specified requirements have been fulfilled
Note 1 to entry: The term "verified" is used to designate the corresponding status.
Note 2 to entry: Confirmation can comprise activities such as performing alternative calculations, comparing a
new design specification with a similar proven design specification, undertaking tests and demonstrations, and
reviewing documents prior to issue.
Note 3 to entry: See Annex B for additional information.
[SOURCE: ISO 9000:2015]
4 General laboratory requirement for microbial pathogens NAAT
4.1 General laboratory risk management and biosafety requirements
The medical laboratory management shall ensure the safety and protection of laboratory staff and
service personnel. The requirements of ISO 15190 apply. As many microbes are pathogenic appropriate
biosafety standards shall be applied.
The nucleic acid-based examination process shall be assessed by the medical laboratory to identify risks
such as failure modes, operation errors, hazards and hazardous situations. The risks to patients and
laboratory workers shall be identified prior to, and mitigated during examination development. Risks
shall also be reviewed, monitored and mitigated prior to and during implementation, and regularly
during the life cycle of operation.
[9]
NOTE 1 The medical laboratory can also refer to ISO 35001 .
[11]
NOTE 2 The WHO biosafety manual can be applied, WHO/CDS/EPR/2006.6 .
NOTE 3 The general principles and risk management practices described in [8] can also be applied to medical
laboratories.
[40]
NOTE 4 For general guidance for reduction of laboratory error, see also ISO 22367 .
NOTE 5 For information about quality control planning based upon risk management principles, see also
[1]
CLSI EP23 .
4.2 General laboratory set ups for pathogen detection
General requirements for best practice in NAAT laboratory set up shall be followed and typically include
separation of pre- and post-amplification rooms and potentially further modularization of laboratory
steps. Specific considerations around pathogen containment shall also be adhered to along with the
appropriate risk assessment.
10 © ISO 2020 – All rights reserved

Of note are situations where sample containment risk level is greater than that required for handling of
extracted nucleic acids. Pathogens of higher risk categories will typically require lower risk category for
handling extracted nucleic acid and NAAT analysis. However, it cannot be assumed that the extraction
procedure will render the sample non-infectious. Therefore, safety risk assessment shall be considered.
To increase biosafety, pathogen inactivation should be achieved at the earliest possible stage. However,
inactivation method needs to be confirmed and risk and safety procedures applied as appropriate.
General Laboratory setup for management and reduction of contamination.
Contamination sources can be grouped into five categories and the laboratory should be setup to
minimize contamination risk from each potential source:
1) Environmental
Derived from outside the laboratory. Not generally a problem for pathogen detection although
where closely related environmental species may cause contamination risk positive pressure
laboratories should be used.
Further detailed information to unidirectional workflow and air pressure conditions are given
in 7.2.2.
2) Laboratory
Major source of contamination as a result of preparation of large amounts of nucleic acid by
the laboratory. This is typically a problem when using NAATs as they function by generating
large amounts of the target (amplicon) in question; this is the predominant source of any NAAT
contamination. Laboratory sources can also be derived from the use of vectors containing target
sequences. In pathogen detection it can also be derived from microbial culture of pathogens of
interest. This can be mitigated by separation of sample preparation and test set-up from other
activities where large amounts of genetic material may be present. Further risk of contamination
should be reduced by ensuring task specific laboratory equipment and coats.
3) Reagents
Derived from the fact that many NAAT reagents are recombinant in source, therefore a low-level
amount of bacterial DNA may persist. This is particularly a problem when targeting orthogonal
genes like 16S ribosomal RNA gene.
4) Analyst
Not usually a major source of contamination for pathogen specific NAAT as analysts not usually
carrying pathogen. Potentially a problem, where pathogen (or closely related species) can exist
asymptomatically. Standard laboratory procedures (e.g. protective clothing, gloves, filter tips etc.)
can mitigate source.
5) Sample
Potential major, and frequently unrecognised, source of contamination. Where high pathogen titre
sample is prepared next to low titre or negative sample and cross contamination is likely. Standard
laboratory procedures (e.g. filter tips) can mitigate source.
Standard operating procedures shall be written, implemented and staff trained to reduce the risk
for contamination. This may include restricted personnel direction and forward workflow from
amplicon negative to amplicon positive areas and/or use of disposable labcoats or other protective
clothing. This may not apply when using closed systems which automate the nucleic acid extraction,
amplification and detection as a single workflow.
Ultimately contamination shall be monitored in an appropriate manner to determine its influence
on results. Laboratory, housekeeping, and all other personnel entering the laboratory, shall be
trained, and the training documented.
For further detailed information see also Clause 5.
4.3 Commercial equipment (including software programs)
Equipment intended to perform nucleic acid amplification-based examinations, including software
programs necessary to perform the analysis, shall be installed, verified, calibrated and maintained
according to the manufacturer’s instructions for use and documented laboratory procedures.
Where applicable, integration of laboratory instruments into existing IT infrastructure shall be verified.
EXAMPLE Connectivity to databases, bioinformatic functions, etc.
If multiple instruments are potentially used for the same nucleic acid test, inter-instrument comparison
shall be performed to ensure comparability of results. The laboratory shall verify any in-house
developed interfaces between instrument components and shall also verify interfaces between
instrument components developed by manufacturer.
4.4 Laboratory personnel
Personnel assigned to perform nucleic acid amplification-based examinations shall be qualified and
trained to the level of competence required for the specific NAAT and pathogen including continuing
education to maintain competency.
The qualification and training of these personnel shall be documented.
5 Planning and implementation of pathogen NAAT assay
In general the criteria of the design are listed in a design plan.
Design and development planning shall include:
1. Definition of user needs and stakeholder requirements
2. Definition of the intended medical use
3. Performance requirements and specifications and other design requirements and specifications
based on the intended use
4. Product risk assessment
5. Assay design and assay component supplier qualification, this should include but is not limited to:
Specimen collection and processing, nucleic acid extraction, nucleic acid amplification, and
detection and identification of nucleic acids of the target microbial pathogen, laboratory design,
work flow and laboratory practices.
6. Performing of the feasibility phase
7. Verification and validation planning
8. Verification of design specifications
EXAMPLES Detection limit, cut-off values, analytical specificity (including cross-reactivity and
interference), precision, carryover, linearity, and where appropriate, calibrator commutability and
traceability of results to reference materials or reference measurement procedures.
9. Laboratory scale production process planning
10. Validation of the intended use
11. Design changes during and after the development of the examination shall be documented
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Pathogen specific considerations should include, but not be limited to:
Pathogen genetic consideration (sequence heterogeneity both within infection and across species,
required operational taxonomic unit (OTU), specificity when considering closely related species,
genetic association when considering resistance genes). Examples are:
— Use of multiplex panels (e.g. respiratory panel)
— Latency, carriage, reference ranges, relevant for multiplex panels which may detect organisms not
part of the differential diagnosis
5.1 Quality control material
5.1.1 Examination of quality control material
For the receipt of reliable data and assay results the selection and
...