ISO 21474-1:2020
(Main)In vitro diagnostic medical devices — Multiplex molecular testing for nucleic acids — Part 1: Terminology and general requirements for nucleic acid quality evaluation
In vitro diagnostic medical devices — Multiplex molecular testing for nucleic acids — Part 1: Terminology and general requirements for nucleic acid quality evaluation
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 — Partie 1: Terminologie et exigences générales pour l’évaluation de la qualité des acides nucléiques
General Information
Standards Content (Sample)
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 2020
© 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
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
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
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.
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.]
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), normal
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.