In vitro diagnostic medical devices -- Multiplex molecular testing for nucleic acids

This document provides the terms and general requirements for the evaluation of the quality of nucleic acids as the analytes for multiplex molecular tests, which simultaneously identify two or more nucleic acid target sequences of interest. This document is applicable to all multiplex molecular methods used for examination using in vitro diagnostic (IVD) medical devices and laboratory developed tests (LDTs). It provides information for both qualitative and quantitative detection of nucleic acid target sequences. This document is intended as guidance for multiplex molecular assays that detect and/or quantify human nucleic acid target sequences or microbial pathogen nucleic acid target sequences from human clinical specimens. This document is applicable to any molecular in vitro diagnostic examination performed by medical laboratories. It is also intended to be used by laboratory customers, in vitro diagnostics developers and manufacturers, biobanks, institutions and commercial organizations performing biomedical research, and regulatory authorities. This document is not applicable to metagenomics. NOTE An examination procedure developed for a laboratory's own use is often referred to as a "laboratory developed test", "LDT", or "in-house test".

Dispositifs médicaux de diagnostic in vitro -- Tests moléculaires multiplex pour les acides nucléiques

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INTERNATIONAL ISO
STANDARD 21474-1
First edition
2020-08
In vitro diagnostic medical devices —
Multiplex molecular testing for
nucleic acids —
Part 1:
Terminology and general
requirements for nucleic acid quality
evaluation
Dispositifs médicaux de diagnostic in vitro — Tests moléculaires
multiplex pour les acides nucléiques —
Partie 1: Terminologie et exigences générales pour l’évaluation de la
qualité des acides nucléiques
Reference number
ISO 21474-1:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO 21474-1:2020(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 21474-1:2020(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 General considerations .................................................................................................................................................................................. 8

4.1 General ........................................................................................................................................................................................................... 8

4.1.1 Pre-analytical phase considerations ............................................................................................................... 8

4.1.2 Specimen quality considerations ....................................................................................................................... 8

4.1.3 Nucleic acid quality considerations ................................................................................................................. 9

4.2 Multiplex molecular test quality nucleic acid and evaluation ........................................................................ 9

4.2.1 Evaluation of nucleic acid quality for multiplex molecular tests ........................................... 9

4.2.2 Evaluation of nucleic acid quantity ...............................................................................................................10

5 Procedure for preparation of nucleic acid..............................................................................................................................10

5.1 General ........................................................................................................................................................................................................10

5.2 Preparation of samples .................................................................................................................................................................11

5.2.1 General...................................................................................................................................................................................11

5.2.2 Consideration on tissue preparation ...........................................................................................................11

5.2.3 Nucleic acid extraction and purification ..................................................................................................12

5.2.4 Quality evaluation method ...................................................................................................................................13

Annex A (informative) Evaluation of RNA Integrity ...........................................................................................................................15

Annex B (informative) Evaluation of DNA Integrity ...........................................................................................................................16

Annex C (informative) Use of PCR to assess amplifiable DNA from FFPE samples ............................................17

Annex D (informative) microRNA Sample ....................................................................................................................................................19

Bibliography .............................................................................................................................................................................................................................20

© ISO 2020 – All rights reserved iii
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ISO 21474-1:2020(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.
A list of all parts in the ISO 21474 series can be found on the ISO website.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2020 – All rights reserved
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ISO 21474-1:2020(E)
Introduction

The first generation of in vitro diagnostics (IVD) medical devices for nucleic acid-based molecular tests

have been focused on detection or quantitation of a single nucleic acid sequence (e.g., viral RNA, mRNA

or genomic DNA) within a clinical specimen. By comparison, a multiplex molecular test simultaneously

measures multiple nucleic acid sequences of interest in a single reaction. The development and clinical

use of multiplex IVD medical devices are rapidly expanding with technological advances and new

elucidation of the clinical significance of many biomarkers.

The measurement of multiple analytes of interest in a clinical specimen is generally performed by the

following successive (or simultaneous) steps. After specimen collection, transport and storage, nucleic

acids are extracted, with or without a subsequent purification procedure. The nucleic acid is then

quantified, and its quality evaluated (if necessary), diluted (if necessary) and subjected to multiplex

molecular test(s). Multiplex molecular tests in current clinical use detect DNA or RNA targets using

various techniques, such as multiplex PCR examinations, microarrays, mass array or massive parallel

sequencing-based methodologies.

Although quality aspects of nucleic acids for single target molecular analysis (such as singleplex PCR)

[1][2]

has been described , this cannot necessarily be applied to multiplex molecular tests. Due to the

inherent competition for more than one nucleic acid target in a multiplex assay, these assays are usually

more sensitive to the isolated nucleic acid quality and quantity than single target assays. The variability

of each specimen in biological, physical and chemical properties can influence the performance of

multiplex assays to a larger degree than single target assays, potentially leading to unreliable results

and hampering patient care. Thus, sample quality evaluation should require additional considerations

for multiplex molecular tests.

The collection, transport and preparation of specimens for medical laboratory use has been addressed

in national and international efforts in general including ISO/TS 20658 “Medical laboratories—

[3]

Requirements for collection, transport, receipt and handling of samples” , “Guideline for the Quality

Management of Specimens for Molecular Methods; The Procurement, Transport, and Preparation of

[4]

Specimens” (Japan, JCCLS) and “Guideline for the Quality Management of Specimens for Molecular

[5]

Methods (Part 2) New Technologies and Sample Quality Control (Japan, JCCLS)” , and more specifically

[6][7][8]

for different biological specimen types in the series of ISO 20166, 20184, and 20186 .

This document describes the terminology and general quality requirements for nucleic acid used in

multiplex molecular tests, in order to ensure reproducible performance of such tests.

NOTE Guidelines, requirements, and performance criteria laid down in this document, are intended to

ensure that comparable, accurate and reproducible results are obtained in different laboratories.

© ISO 2020 – All rights reserved v
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INTERNATIONAL STANDARD ISO 21474-1:2020(E)
In vitro diagnostic medical devices — Multiplex molecular
testing for nucleic acids —
Part 1:
Terminology and general requirements for nucleic acid
quality evaluation
1 Scope

This document provides the terms and general requirements for the evaluation of the quality of nucleic

acids as the analytes for multiplex molecular tests, which simultaneously identify two or more nucleic

acid target sequences of interest. This document is applicable to all multiplex molecular methods used

for examination using in vitro diagnostic (IVD) medical devices and laboratory developed tests (LDTs).

It provides information for both qualitative and quantitative detection of nucleic acid target sequences.

This document is intended as guidance for multiplex molecular assays that detect and/or quantify human

nucleic acid target sequences or microbial pathogen nucleic acid target sequences from human clinical

specimens. This document is applicable to any molecular in vitro diagnostic examination performed

by medical laboratories. It is also intended to be used by laboratory customers, in vitro diagnostics

developers and manufacturers, biobanks, institutions and commercial organizations performing

biomedical research, and regulatory authorities. This document is not applicable to metagenomics.

NOTE An examination procedure developed for a laboratory’s own use is often referred to as a “laboratory

developed test”, “LDT”, or “in-house test”.
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 15189:2012, Medical laboratories — Requirements for quality and competence
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 http:// www .iso .org/ obp

— I EC E le c t r op e d i a : av a i l able at ht t p:// w w w . ele c t r op e d i a ./ or g

3.1
accuracy

closeness of agreement between a measured quantity value and a true quantity value of a measurand

Note 1 to entry: The term accuracy, when applied to a set of test results, involves a combination of random

components and a common systematic error or bias component (ISO 3534-2:2006, 3.3.1).

[SOURCE: ISO/IEC Guide 99:2007, 2.13, modified — “NOTE 1”, “NOTE 2” and “NOTE 3” have been deleted,

and new “Note 1 to entry” has been added.]
© ISO 2020 – All rights reserved 1
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ISO 21474-1:2020(E)
3.2
algorithm

set of rules or calculations applied to test data that generate an interpretable or reportable result

3.3
allele

any of several forms of a gene that is responsible for hereditary variation

Note 1 to entry: An allele can also be defined as:

1) one of the alternate forms of a polymorphic DNA sequence that is not necessarily contained within a gene;

2) one of the alternative forms of a gene that may occupy a given locus.
3.4
allelic ratio

ratio of a specified allele (3.3) to the total number of alleles (3.3), normally expressed as a fraction

Note 1 to entry: For example, if a specific allele (3.3) represents 40 % of the total alleles (3.3) found at a given

locus, the allelic ratio is 0,4.
Note 2 to entry: Allelic ratio is synonymous with allele frequency.
3.5
analyte
component represented in the name of a measurable quantity
[SOURCE: ISO 17511:2020, 3.1, modified — The example has been deleted.]
3.6
chemical purity

degree of contamination with chemical substances that influences the multiplex analysis

Note 1 to entry: The purity of nucleic acid for PCR is absence of interfering organic and protein components

carried through from the extraction step, as well as contaminating nucleic acids.

3.7
DNA microarray
DNA chip

solid substrate where a collection of probe DNA arranged in a specific design is attached in a high-

density fashion directly or indirectly, that assays large amounts of biological material using high-

throughput screening methods
[SOURCE: ISO 16578: 2013, 3.3]
3.8
documented procedure

specified way to carry out an activity or a process that is documented, implemented and maintained

interlaboratory comparison (3.13)
3.9
evaluation method
method of evaluating the quality specified for nucleic acid
3.10
expiry date
expiration date

upper limit of the time interval during which the performance characteristics of a material stored

under specified conditions can be assured

Note 1 to entry: Expiry dates are assigned to IVD reagents (3.16), calibrators, control materials and other

components by the manufacturer based on experimentally determined stability (3.38) properties.

2 © ISO 2020 – All rights reserved
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ISO 21474-1:2020(E)

[SOURCE: ISO 18113-1:2009, 3.17, modified — “Note 2 to entry” and “Note 3 to entry” have been deleted.]

3.11
external measurement standard
reference standard

material or substrate prepared for testing the compatibility of the methods of multiplex analysis, whose

property value is derived as a consensus value based on collaborative experimental work under the

auspices of a scientific or engineering group
Note 1 to entry: This is commonly targeted at the multiplex molecular analysis.

Note 2 to entry: Reference material can be used as an alternative of external measurement standard.

[SOURCE: ISO 16578:2013, 3.9, modified — “Note 1 to entry” and “Note 2 to entry” have been added.]

3.12
intended use
intended purpose

objective intent of an IVD manufacturer regarding the use of a product, process or service as reflected

in the specifications, instructions and information supplied by the IVD manufacturer

[SOURCE: ISO 18113-1:2009, 3.31, modified — “Note 1 to entry” and “Note 2 to entry” have been

deleted.]
3.13
interlaboratory comparison

organization, performance and evaluation of measurements or tests on the same or similar items by

two or more laboratories in accordance with predetermined conditions
[SOURCE: ISO/IEC 17043:2010, 3.4]
3.14
in vitro diagnostic instrument
IVD instrument

equipment or apparatus intended by a manufacturer to be used as an IVD medical device (3.15)

[SOURCE: ISO 18113-1:2009, 3.26, modified — “Note 1 to entry” has been deleted.]
3.15
in vitro diagnostic product
in vitro diagnostic medical device
IVD medical device

reagents, instruments, and systems intended for use in the diagnosis of disease or other conditions,

including a determination of the state of health, in order to cure, mitigate, treat, or prevent disease or

its sequelae
[SOURCE: 21CFR809.3 of the US Federal Food, Drug and Cosmetic Act]
3.16
in vitro diagnostic reagent
IVD reagent

chemical, biological, or immunological components, solutions or preparations intended by the

manufacturer to be used with an IVD medical device (3.15)
[SOURCE: ISO 18113-1:2009, 3.28, modified — “Note 1 to entry” has been deleted.]
© ISO 2020 – All rights reserved 3
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ISO 21474-1:2020(E)
3.17
laboratory developed tests
LDTs

type of in vitro diagnostic devices that are intended for clinical use and are designed, manufactured

and used within a single laboratory
Note 1 to entry: It is often referred to as a “in-house test”.
[SOURCE: CLSI QSRLDT]
3.18
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: IUPAC recommends default values for α and β equal to 0,05.

Note 2 to entry: This is for LODs when the tests are evaluating the presence or absence of multiple analytes (3.5)

rather than a multivariable molecular test (3.26).

Note 3 to entry: Limit of detection, LOD, is alternatively defined as 1) the lowest quantity of a nucleic acid that

can be sequenced reliably and distinguished from its absence typically within a stated confidence limit; 2) the

minimum detectable allelic fraction in a given sample.
[SOURCE: CLSI MM09 2014]
3.19
limit of detection for microarray platform
limit of detection for multiplex molecular test platform
LODP

lowest relative quantity of the external measurement standard (3.11) (or reference material) that

can be consistently detected experimentally at a 95 % confidence level, given a known (determined/

estimated) number of copies and/or concentration of the external measurement standard (3.11) (or

reference material)
Note 1 to entry: This is commonly targeted at the multiplex molecular analysis.

Note 2 to entry: LODP can be used as a performance indicator replaced by limit of detection (3.18) for multiplex

analysis.

[SOURCE: ISO 16578:2013, 3.1, modified — “Note 1 to entry” and “Note 2 to entry” have been added.]

3.20
massive parallel sequencing

methodology that enables high-throughput DNA sequencing using the concept of processing a very

large number of molecules in parallel

Note 1 to entry: For example but not limited to the technologies with miniaturized and parallelized platforms

for sequencing of thousands to millions of short reads (≈50 to 400 bases), or polymerase-based real-time DNA

sequencing platform enabling long read (mean length ≈10,000–15,000 bases).
3.21
microRNA

17 to 25 nucleotide-long single strand RNA relating to post transcriptional expression regulation

3.22
multiple sequences of analyte(s)

constituent of a sample with multiple sequences of nucleic acid measured simultaneously

Note 1 to entry: This includes extracted nucleic acid and that before and/or after amplification in case of nucleic

acid amplification-based assay.
4 © ISO 2020 – All rights reserved
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ISO 21474-1:2020(E)
3.23
multiplex molecular test

in vitro diagnostic test that simultaneously evaluates sequence identity and/or amounts of multiple,

namely two or more nucleic acid targets of interest in a single run of the assay, such as multiplex PCR

(3.25), multiple hybridization detection, microarray and massive parallel sequencing (3.20) based

methodologies

Note 1 to entry: “Multiplex” is defined as “those in which two or more targets are simultaneously detected

through a common process of sample preparation, target or signal amplification, allele (3.3) discrimination, and

[24]
collective interpretation. (CLSI/MM17-A ).

Note 2 to entry: Targets of interest is defined as detection targets of interest and exclude the control material

from being a target.
3.24
multiplex molecular test quality nucleic acid

nucleic acid template with appropriate property that ensures the measurement by a multiplex molecular

test (3.23) such as that of sufficient length, quantity, chemical purity (3.6), structural integrity (3.40),

and presence of nucleic acid sequence of interest
3.25
multiplex PCR

PCR technique that employs multiple pairs of primers combined within a single reaction mixture to

produce multiple amplicons simultaneously
[SOURCE: ISO 16577:2016, 3.117]
3.26
multivariable molecular test

molecular test that combines the values of multiple variables using an interpretation function to yield a

single, patient-specific result including “classification,” “score” and/or “index”

Note 1 to entry: This is usually based on a platform of multiplex molecular tests.

Note 2 to entry: This is intended for use in the diagnosis of disease or other conditions, or in the cure, mitigation,

treatment or prevention of disease.

Note 3 to entry: The term “multivariable” as used in statistics implies the evaluation of multiple outcomes rather

than using multiple variables to evaluate a single outcome.
3.27
pathogen
infectious agent that causes diseases in its host

Note 1 to entry: Pathogen includes some virus, viroid, prion, bacterium, fungus, or parasite.

[SOURCE: ISO 15714:2019, 3.1.2, modified.]
3.28
PCR quality DNA

DNA template of sufficient length, quantity, chemical purity (3.6), and structural integrity (3.40) to be

amplified by PCR
[SOURCE: ISO 24276:2006, 3.2.3, modified — “quantity” is added.]
© ISO 2020 – All rights reserved 5
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ISO 21474-1:2020(E)
3.29
preanalytical phase
pre-examination processes

processes that start, in chronological order, from the clinician’s request and include the examination

request, preparation and identification of the patient, collection of the primary sample(s), and

transportation to and within the laboratory, isolation of analytes, and end when the analytical

examination begins

[SOURCE: ISO 15189:2012, 3.15, modified — The words "isolation of analytes" have been added.]

3.30
primary sample
specimen

discrete portion of a body fluid or tissue taken for examination, study or analysis of one or more

quantities or properties assumed to apply for the whole

Note 1 to entry: Global Harmonisation Task Force (GHTF) uses the term specimen in its harmonized guidance

documents to mean a sample of biological origin intended for examination by a medical laboratory.

Note 2 to entry: In some ISO and CEN documents, a specimen is defined as “a biological sample derived from the

human body".

Note 3 to entry: In some countries, the term “specimen” is used instead of primary sample (or a subsample

of it), which is the sample prepared for sending to, or as received by, the laboratory and which is intended for

examination.
[SOURCE: ISO 15189:2012, 3.16]
3.31
range of reliable signal

ability (within a given range) to provide results that are directly proportional to the concentration and/

or copy number of the external measurement standard (3.11) (or reference material)

Note 1 to entry: This is used mostly for quantitative but not qualitative tests.
Note 2 to entry: Linear range or analytical measurable range is also used.

[SOURCE: ISO 16578:2013, 3.2, modified — "Note 1 to entry” and “Note 2 to entry” have been added.]

3.32
reportable range

region of the genome in which sequence of an acceptable quality can be covered by the laboratory test

Note 1 to entry: The reportable range is also defined as “the range of test values over which the relationship

between the instrument, kit, or system's measurement response is shown to be valid” (US CFR 493).

3.33
reference range

reportable sequence variations the assay can detect that are expected to occur in an unaffected

population

Note 1 to entry: A reference range is also defined as a set of values that include upper and lower limits of a

laboratory test based on a group of otherwise healthy people.
3.34
reverse transcription

synthesis of DNA from an RNA template using a reverse transcriptase enzyme combined with an RT-

primer in the presence of deoxyribonucleoside triphosphate
[SOURCE: ISO 22174:2005, 3.3.1]
6 © ISO 2020 – All rights reserved
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ISO 21474-1:2020(E)
3.35
RT-PCR

method consisting of two reactions, a reverse transcription (3.34) of RNA to DNA and a subsequent PCR

[SOURCE: ISO 22174:2005, 3.4.2]
3.36
RT-PCR quality RNA

RNA template of sufficient length, quantity, chemical purity and structural integrity suitable for reverse

transcription (3.34) and PCR
[SOURCE: ISO 22174:2005, 3.2.4, modified.]
3.37
sample
one or more parts taken from a primary sample
[SOURCE: ISO 15189:2012, 3.24, modified — The example has been deleted.]
3.38
stability

ability of an IVD medical device (3.15) to maintain its performance characteristics within the limits

specified by the manufacturer
Note 1 to entry: Stability applies to:

— IVD reagents (3.16), calibrators and controls, when stored, transported and used in the conditions specified

by the manufacturer;

— Reconstituted lyophilized materials, working solutions and materials removed from sealed containers

(when prepared, used and stored according to the manufacturer’s instructions for use).

Note 2 to entry: Stability of an IVD reagent (3.16) or measuring system is normally quantified with respect to time:

— in terms of the duration of a time interval over which a metrological property changes by a stated amount;

— in terms of the change of a property over a stated time interval.

[SOURCE: ISO 18113-1:2009, 3.68, modified — “Measuring instruments or measuring systems after

calibration “in the “Note 1 to entry” and “Note 3 to entry” have been deleted.]
3.39
specimen stability
resistance of a specimen to quality change during long-term storage

[SOURCE: ISO 23833: 2013, 5.5.10, modified — The text “changes in chemical composition during

electron bombardment, i.e. the resistance to change of the intensity of the relevant characteristic X

rays observed during the time the specimen is exposed to the electron beam” has been replaced with

“quality change during long-term storage”.]
3.40
structural integrity
degree of preservation of nucleic acid reflecting the original state
3.41
validation

confirmation, through the provision of objective evidence, that the requirements for a specific intended

use (3.12) or application have been fulfilled

Note 1 to entry: The word “validated” is used to designate the corresponding status.

[SOURCE: ISO 9000:2015, 3.8.13, modified — “Note 1 to entry” and “Note 3 to entry” have been deleted.]

© ISO 2020 – All rights reserved 7
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ISO 21474-1:2020(E)
3.42
verification

confirmation, through provision of objective evidence, that specified requirements have been fulfilled

Note 1 to entry: The word “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.

[SOURCE: ISO 9000:2015, 3.8.12, modified — “Note 1 to entry” and “Note 2 to entry” have been

reworded.]
4 General considerations
4.1 General
4.1.1 Pre-analytical phase considerations
For general statements on quality management s
...

FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 21474-1
ISO/TC 212
In vitro diagnostic medical devices —
Secretariat: ANSI
Multiplex molecular testing for
Voting begins on:
2020-05-11 nucleic acids —
Voting terminates on:
Part 1:
2020-07-06
Terminology and general
requirements for nucleic acid quality
evaluation
Dispositifs médicaux de diagnostic in vitro — Tests moléculaires
multiplex pour les acides nucléiques —
Partie 1: Terminologie et exigences générales pour l’évaluation de la
qualité des acides nucléiques
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 SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/FDIS 21474-1:2020(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN-
DARDS TO WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS. ISO 2020
---------------------- Page: 1 ----------------------
ISO/FDIS 21474-1:2020(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
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/FDIS 21474-1:2020(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 General considerations .................................................................................................................................................................................. 8

4.1 General ........................................................................................................................................................................................................... 8

4.1.1 Pre-analytical phase considerations ............................................................................................................... 8

4.1.2 Specimen quality considerations ....................................................................................................................... 8

4.1.3 Nucleic acid quality considerations ................................................................................................................. 9

4.2 Multiplex molecular test quality nucleic acid and evaluation ........................................................................ 9

4.2.1 Evaluation of nucleic acid quality for multiplex molecular tests ........................................... 9

4.2.2 Evaluation of nucleic acid quantity ...............................................................................................................10

5 Procedure for preparation of nucleic acid..............................................................................................................................10

5.1 General ........................................................................................................................................................................................................10

5.2 Preparation of samples .................................................................................................................................................................11

5.2.1 General...................................................................................................................................................................................11

5.2.2 Consideration on tissue preparation ...........................................................................................................11

5.2.3 Nucleic acid extraction and purification ..................................................................................................12

5.2.4 Quality evaluation method ...................................................................................................................................13

Annex A (informative) Evaluation of RNA Integrity ...........................................................................................................................15

Annex B (informative) Evaluation of DNA Integrity ...........................................................................................................................16

Annex C (informative) Use of PCR to assess amplifiable DNA from FFPE samples ............................................17

Annex D (informative) microRNA Sample ....................................................................................................................................................19

Bibliography .............................................................................................................................................................................................................................20

© ISO 2020 – All rights reserved iii
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ISO/FDIS 21474-1:2020(E)
Foreword

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This document was prepared by Technical Committee ISO/TC 212, Clinical laboratory testing and in

vitro diagnostic test systems.
A list of all parts in the ISO 21474 series can be found on the ISO website.

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ISO/FDIS 21474-1:2020(E)
Introduction

The first generation of in vitro diagnostics (IVD) medical devices for nucleic acid-based molecular tests

have been focused on detection or quantitation of a single nucleic acid sequence (e.g., viral RNA, mRNA

or genomic DNA) within a clinical specimen. By comparison, a multiplex molecular test simultaneously

measures multiple nucleic acid sequences of interest in a single reaction. The development and clinical

use of multiplex IVD medical devices are rapidly expanding with technological advances and new

elucidation of the clinical significance of many biomarkers.

The measurement of multiple analytes of interest in a clinical specimen is generally performed by the

following successive (or simultaneous) steps. After specimen collection, transport and storage, nucleic

acids are extracted, with or without a subsequent purification procedure. The nucleic acid is then

quantified, and its quality evaluated (if necessary), diluted (if necessary) and subjected to multiplex

molecular test(s). Multiplex molecular tests in current clinical use detect DNA or RNA targets using

various techniques, such as multiplex PCR examinations, microarrays, mass array or massive parallel

sequencing-based methodologies.

Although quality aspects of nucleic acids for single target molecular analysis (such as singleplex PCR)

[1][2]

has been described , this cannot necessarily be applied to multiplex molecular tests. Due to the

inherent competition for more than one nucleic acid target in a multiplex assay, these assays are usually

more sensitive to the isolated nucleic acid quality and quantity than single target assays. The variability

of each specimen in biological, physical and chemical properties can influence the performance of

multiplex assays to a larger degree than single target assays, potentially leading to unreliable results

and hampering patient care. Thus, sample quality evaluation should require additional considerations

for multiplex molecular tests.

The collection, transport and preparation of specimens for medical laboratory use has been addressed

in national and international efforts in general including ISO/TS 20658 “Medical laboratories—

[3]

Requirements for collection, transport, receipt and handling of samples” , “Guideline for the Quality

Management of Specimens for Molecular Methods; The Procurement, Transport, and Preparation of

[4]

Specimens” (Japan, JCCLS) and “Guideline for the Quality Management of Specimens for Molecular

[5]

Methods (Part 2) New Technologies and Sample Quality Control (Japan, JCCLS)” , and more specifically

[6][7][8]

for different biological specimen types in the series of ISO 20166, 20184, and 20186 .

This document describes the terminology and general quality requirements for nucleic acid used in

multiplex molecular tests, in order to ensure reproducible performance of such tests.

NOTE Guidelines, requirements, and performance criteria laid down in this document, are intended to

ensure that comparable, accurate and reproducible results are obtained in different laboratories.

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FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 21474-1:2020(E)
In vitro diagnostic medical devices — Multiplex molecular
testing for nucleic acids —
Part 1:
Terminology and general requirements for nucleic acid
quality evaluation
1 Scope

This document provides the terms and general requirements for the evaluation of the quality of nucleic

acids as the analytes for multiplex molecular tests, which simultaneously identify two or more nucleic

acid target sequences of interest. This document is applicable to all multiplex molecular methods used

for examination using in vitro diagnostic (IVD) medical devices and laboratory developed tests (LDTs).

It provides information for both qualitative and quantitative detection of nucleic acid target sequences.

This document is intended as guidance for multiplex molecular assays that detect and/or quantify human

nucleic acid target sequences or microbial pathogen nucleic acid target sequences from human clinical

specimens. This document is applicable to any molecular in vitro diagnostic examination performed

by medical laboratories. It is also intended to be used by laboratory customers, in vitro diagnostics

developers and manufacturers, biobanks, institutions and commercial organizations performing

biomedical research, and regulatory authorities. This document is not applicable to metagenomics.

NOTE An examination procedure developed for a laboratory’s own use is often referred to as a “laboratory

developed test”, “LDT”, or “in-house test”.
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 15189:2012, Medical laboratories — Requirements for quality and competence
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 http:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia ./ org
3.1
accuracy

closeness of agreement between a measured quantity value and a true quantity value of a measurand

Note 1 to entry: The term accuracy, when applied to a set of test results, involves a combination of random

components and a common systematic error or bias component (ISO 3534-2:2006, 3.3.1).

[SOURCE: ISO/IEC Guide 99:2007, 2.13, modified — “NOTE 1”, “NOTE 2” and “NOTE 3” have been deleted,

and new “Note 1 to entry” has been added.]
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ISO/FDIS 21474-1:2020(E)
3.2
algorithm

a set of rules or calculations applied to test data that generate an interpretable or reportable result

3.3
allele

any of several forms of a gene that is responsible for hereditary variation

Note 1 to entry: An allele can also be defined as:

Note 2 to entry: 1) one of the alternate forms of a polymorphic DNA sequence that is not necessarily contained

within a gene;

Note 3 to entry: 2) one of the alternative forms of a gene that may occupy a given locus.

3.4
allelic ratio

the ratio of a specified allele (3.3) to the total number of alleles (3.3), normally expressed as a fraction

Note 1 to entry: For example, if a specific allele (3.3) represents 40 % of the total alleles (3.3) found at a given

locus, the allelic ratio is 0,4.
Note 2 to entry: Allelic ratio is synonymous with allele frequency.
3.5
analyte
component represented in the name of a measurable quantity
[SOURCE: ISO 17511:2003, 3.2, modified — The example has been deleted.]
3.6
chemical purity

degree of contamination with chemical substances that influences the multiplex analysis

Note 1 to entry: The purity of nucleic acid for PCR is absence of interfering organic and protein components

carried through from the extraction step, as well as contaminating nucleic acids.

3.7
DNA microarray
DNA chip

solid substrate where a collection of probe DNA arranged in a specific design is attached in a high-

density fashion directly or indirectly, that assays large amounts of biological material using high-

throughput screening methods
[SOURCE: ISO 16578: 2013, 3.3]
3.8
documented procedure

specified way to carry out an activity or a process that is documented, implemented and maintained

interlaboratory comparison (3.12)
3.9
evaluation method
method of evaluating the quality specified for nucleic acid
3.10
expiry date
expiration date

upper limit of the time interval during which the performance characteristics of a material stored

under specified conditions can be assured

Note 1 to entry: Expiry dates are assigned to IVD reagents (3.15), calibrators, control materials and other

components by the manufacturer based on experimentally determined stability (3.35) properties.

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ISO/FDIS 21474-1:2020(E)

[SOURCE: ISO 18113-1:2009, 3.17, modified — “Note 2 to entry” and “Note 3 to entry” have been deleted.]

3.11
external measurement standard
reference standard

material or substrate prepared for testing the compatibility of the methods of multiplex analysis, whose

property value is derived as a consensus value based on collaborative experimental work under the

auspices of a scientific or engineering group
Note 1 to entry: This is commonly targeted at the multiplex molecular analysis.

Note 2 to entry: Reference material can be used as an alternative of external measurement standard.

[SOURCE: ISO 16578:2013, 3.9, modified — “Note 1 to entry” and “Note 2 to entry” have been added.]

3.12
intended use
intended purpose

objective intent of an IVD manufacturer regarding the use of a product, process or service as reflected

in the specifications, instructions and information supplied by the IVD manufacturer

[SOURCE: ISO 18113-1:2009, 3.31, modified — “Note 1 to entry” and “Note 2 to entry” have been

deleted.]
3.13
interlaboratory comparison

organization, performance and evaluation of measurements or tests on the same or similar items by

two or more laboratories in accordance with predetermined conditions
[SOURCE: ISO/IEC 17043:2010, 3.4]
3.14
in vitro diagnostic instrument
IVD instrument

equipment or apparatus intended by a manufacturer to be used as an IVD medical device (3.14)

[SOURCE: ISO 18113-1:2009, 3.26, modified — “Note 1 to entry” has been deleted.]
3.15
in vitro diagnostic product
in vitro diagnostic medical device
IVD medical device

reagents, instruments, and systems intended for use in the diagnosis of disease or other conditions,

including a determination of the state of health, in order to cure, mitigate, treat, or prevent disease or

its sequelae
[SOURCE: 21CFR809.3 of the US Federal Food, Drug and Cosmetic Act]
3.16
in vitro diagnostic reagent
IVD reagent

chemical, biological, or immunological components, solutions or preparations intended by the

manufacturer to be used with an IVD medical device (3.14)
[SOURCE: ISO 18113-1:2009, 3.28, modified — “Note 1 to entry” has been deleted.]
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ISO/FDIS 21474-1:2020(E)
3.17
laboratory developed tests
LDTs

type of in vitro diagnostic devices that are intended for clinical use and are designed, manufactured

and used within a single laboratory
Note 1 to entry: It is often referred to as a “in-house test”.
[SOURCE: CLSI QSRLDT]
3.18
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: IUPAC recommends default values for α and β equal to 0,05.

Note 2 to entry: This is for LODs when the tests are evaluating the presence or absence of multiple analytes (3.5)

rather than a multivariable molecular test (3.25).

Note 3 to entry: Limit of detection, LOD, is alternatively defined as 1) the lowest quantity of a nucleic acid that

can be sequenced reliably and distinguished from its absence typically within a stated confidence limit; 2) the

minimum detectable allelic fraction in a given sample.
[SOURCE: CLSI MM09 2014]
3.19
limit of detection for microarray platform
limit of detection for multiplex molecular test platform
LODP

lowest relative quantity of the external measurement standard (3.10) (or reference material) that

can be consistently detected experimentally at a 95 % confidence level, given a known (determined/

estimated) number of copies and/or concentration of the external measurement standard (3.10) (or

reference material)
Note 1 to entry: This is commonly targeted at the multiplex molecular analysis.

Note 2 to entry: LODP can be used as a performance indicator replaced by limit of detection (3.18) for multiplex

analysis.

[SOURCE: ISO 16578:2013, 3.1, modified — “Note 1 to entry” and “Note 2 to entry” have been added.]

3.20
massive parallel sequencing

methodology that enables high-throughput DNA sequencing using the concept of processing a very

large number of molecules in parallel

Note 1 to entry: For example but not limited to the technologies with miniaturized and parallelized platforms

for sequencing of thousands to millions of short reads (≈50 to 400 bases), or polymerase-based real-time DNA

sequencing platform enabling long read (mean length ≈10,000–15,000 bases).
3.21
microRNA

17 to 25 nucleotide-long single strand RNA relating to post transcriptional expression regulation

3.22
multiple sequences of analyte(s)

constituent of a sample with multiple sequences of nucleic acid measured simultaneously

Note 1 to entry: This includes extracted nucleic acid and that before and/or after amplification in case of nucleic

acid amplification-based assay.
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ISO/FDIS 21474-1:2020(E)
3.23
multiplex molecular test

in vitro diagnostic test that simultaneously evaluates sequence identity and/or amounts of multiple,

namely two or more nucleic acid targets of interest in a single run of the assay, such as multiplex PCR

(3.24), multiple hybridization detection, microarray and massive parallel sequencing (3.19) based

methodologies

Note 1 to entry: “Multiplex” is defined as “those in which two or more targets are simultaneously detected

through a common process of sample preparation, target or signal amplification, allele (3.3) discrimination, and

[12]
collective interpretation. (CLSI/MM17-A ).

Note 2 to entry: Targets of interest is defined as detection targets of interest and exclude the control material

from being a target.
3.24
multiplex molecular test quality nucleic acid

nucleic acid template with appropriate property that ensures the measurement by a multiplex molecular

test (3.22) such as that of sufficient length, quantity, chemical purity (3.6), structural integrity (3.36),

and presence of nucleic acid sequence of interest
3.25
multiplex PCR

PCR technique that employs multiple pairs of primers combined within a single reaction mixture to

produce multiple amplicons simultaneously
[SOURCE: ISO 16577:2016, 3.117]
3.26
multivariable molecular test

molecular test that combines the values of multiple variables using an interpretation function to yield a

single, patient-specific result including “classification,” “score” and/or “index”

Note 1 to entry: This is usually based on a platform of multiplex molecular tests.

Note 2 to entry: This is intended for use in the diagnosis of disease or other conditions, or in the cure, mitigation,

treatment or prevention of disease.

Note 3 to entry: The term “multivariable” as used in statistics implies the evaluation of multiple outcomes rather

than using multiple variables to evaluate a single outcome.
3.27
pathogen
infectious agent that causes diseases in its host

Note 1 to entry: Pathogen includes some virus, viroid, prion, bacterium, fungus, or parasite.

[SOURCE: ISO 15714:2019, 3.1.2, modified.]
3.28
PCR quality DNA

DNA template of sufficient length, quantity, chemical purity (3.6), and structural integrity (3.36) to be

amplified by PCR
[SOURCE: ISO 24276:2006, 3.2.3, modified — “quantity” is added.]
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ISO/FDIS 21474-1:2020(E)
3.29
preanalytical phase
pre-examination processes

processes that start, in chronological order, from the clinician’s request and include the examination

request, preparation and identification of the patient, collection of the primary sample(s), and

transportation to and within the laboratory, isolation of analytes, and end when the analytical

examination begins
[SOURCE: ISO 15189:2012, 3.15, modified.]
3.30
primary sample
specimen

discrete portion of a body fluid or tissue taken for examination, study or analysis of one or more

quantities or properties assumed to apply for the whole

Note 1 to entry: Global Harmonisation Task Force (GHTF) uses the term specimen in its harmonized guidance

documents to mean a sample of biological origin intended for examination by a medical laboratory.

Note 2 to entry: In some ISO and CEN documents, a specimen is defined as “a biological sample derived from the

human body.

Note 3 to entry: In some countries, the term “specimen” is used instead of primary sample (or a subsample

of it), which is the sample prepared for sending to, or as received by, the laboratory and which is intended for

examination.
[SOURCE: ISO 15189:2012, 3.16]
3.31
range of reliable signal

ability (within a given range) to provide results that are directly proportional to the concentration and/

or copy number of the external measurement standard (3.10) (or reference material)

Note 1 to entry: This is used mostly for quantitative but not qualitative tests.
Note 2 to entry: Linear range or analytical measurable range is also used.

[SOURCE: ISO 16578:2013, 3.2, modified –“Note 1 to entry” and “Note 2 to entry” have been added.]

3.32
reportable range

region of the genome in which sequence of an acceptable quality can be covered by the laboratory test

Note 1 to entry: The reportable range is also defined as “the range of test values over which the relationship

between the instrument, kit, or system's measurement response is shown to be valid” (US CFR 493).

3.33
reference range

reportable sequence variations the assay can detect that are expected to occur in an unaffected

population

Note 1 to entry: A reference range is also defined as a set of values that include upper and lower limits of a

laboratory test based on a group of otherwise healthy people.
3.34
reverse transcription

synthesis of DNA from an RNA template using a reverse transcriptase enzyme combined with an RT-

primer in the presence of deoxyribonucleoside triphosphate
[SOURCE: ISO 22174:2005, 3.3.1]
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3.35
RT-PCR

method consisting of two reactions, a reverse transcription (RT) (3.32) of RNA to DNA and a

subsequent PCR
[SOURCE: ISO 22174:2005, 3.4.2]
3.36
RT-PCR quality RNA

RNA template of sufficient length, quantity, chemical purity and structural integrity suitable for reverse

transcription (3.32) and PCR
[SOURCE: ISO 22174:2005, 3.2.4, modified.]
3.37
sample
one or more parts taken from a primary sample
[SOURCE: ISO 15189:2012, 3.24, modified — The example has been deleted.]
3.38
stability

ability of an IVD medical device (3.14) to maintain its performance characteristics within the limits

specified by the manufacturer
Note 1 to entry: Stability applies to:

— IVD reagents (3.15), calibrators and controls, when stored, transported and used in the conditions specified

by the manufacturer;

— Reconstituted lyophilized materials, working solutions and materials removed from sealed containers

(when prepared, used and stored according to the manufacturer’s instructions for use).

Note 2 to entry: Stability of an IVD reagent (3.15) or measuring system is normally quantified with respect to time:

— in terms of the duration of a time interval over which a metrological property changes by a stated amount;

— in terms of the change of a property over a stated time interval.

[SOURCE: ISO 18113-1:2009, 3.68, modified — “Measuring instruments or measuring systems after

calibration “in the “Note 1 to entry” and “Note 3 to entry” have been deleted.]
3.39
specimen stability
resistance of a specimen to quality change during long-term storage
[SOURCE: ISO 23833: 2013, 5.5.10, modified.]
3.40
structural integrity
degree of preservation of nucleic acid reflecting the original state
3.41
validation

confirmation, through the provision of objective evidence, that the requirements for a specific intended

use (3.11) or application have been fulfilled

Note 1 to entry: The word “validated” is used to designate the corresponding status.

[SOURCE: ISO 9000:2015, 3.8.13, modified — “Note 1 to entry” and “Note 3 to entry” have been deleted.]

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3.42
verification

confirmation, through provision of objective evidence, that specified requirements have been fulfilled

Note 1 to entry: The word “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 de
...

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