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
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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 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
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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
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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), 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
[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.]
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
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.]
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
RT
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
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.]
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 systems of medical laboratory and in particular
[3]
on specimen collection and handling, see ISO 15189:2012, 4.2, 5.4.4, 5.4.7 and ISO/TS 20658 . The
requirements on laboratory equipment, reagents, and consumables according to ISO 15189:2012, 5.3
shall be followed; ISO 15189:2012, 5.5.1.2 and 5.5.1.3 can also apply.
The pre-analytical phase generally consists of the following workflow:
— Sample collection, storage and transport;
— Pre-treatment of the sample;
— Nucleic acid extraction and purification.
These pre-analytical factors greatly impact the quality of a sample and subsequent test results. Details
[6][7][8][25][26][27][28][29][30]
for pre-analytic aspects relevant to molecular assays have been described .
One of the major pre-analytical variables causing a strong impact on analytical test results is post-
collection changes of analyte profile (RNA, DNA) by biological causes, such as gene inductions, gene
down regulations, apoptosis etc. These effects for example depend on the duration of warm (warm
ischemia) and cold ischemia and the ambient temperature before formalin fixation. Such usually remain
[6]
unrecognized in analytical tests but are a major source for wrong or unreliable analytical test results
[7][8][30]
. Thus, the transport of the tissue before storage or fixation in formalin should be made in the
shortest time and possibly at low temperature or under vacuum.
Multiplex molecular tests are IVD tests or medical devices that measure two or more nucleic acid
sequence of interest simultaneously. A sample with a high degree of quality should be procured for
multiplex molecular tests to ensure it is fit for purpose.
4.1.2 Specimen quality considerations
Specimen source, collection, preparation, extraction, and purification should be validated and verified
(if necessary) in order to ensure nucleic acid quality is appropriate for all analytes or targets to be
detected within the assay. A panel of targets for multiplex assays includes both high and low prevalence
targets.
8 © ISO 2020 – All rights reserved
The variability of the biological, physical and chemical properties in each specimen can influence the
quality of the nucleic acid obtained, and thus the performance of multiplex molecular assays between
different specimens. However, it is not practical to evaluate all such influences in each specimen. Thus,
the laboratory should ensure that each clinical specimen is procured in a manner so as to avoid such
variability in properties. When it cannot be avoided, the influence of variability in properties should be
evaluated by an appropriate method, such as including an assessment of internal controls in the assay.
4.1.3 Nucleic acid quality considerations
Multiplex molecular test quality nucleic acid is defined as a nucleic acid template with appropriate
properties that ensures measurement by a multiplex molecular test. While general considerations
on nucleic acid quality evaluation are shared by singleplex and multiplex tests, there are unique
considerations for multiplex molecular tests due to the potential for interference or interactions
between the multiple targets and/or test components.
The quality of isolated nucleic acid from a specimen depends on a number of factors including, but
not limited to, the quantity, the chemical purity, the length and the structural integrity of the nucleic
acids, and the abundance of the target of interest. Its influence should be considered in regard to assay
parameters such as LOD and linear range of each analytical method.
The quality of nucleic acid for multiplex molecular test should be evaluated by appropriate methods,
based on the measurement method to be used. Quality evaluations should define the representative
profile of isolated nucleic acid as determined by length, quantity, chemical purity, and structural
integrity, or the detection or quantity of the representative genes (for example, internal control genes).
Process control materials should be used to monitor both the pre-analytical and analytical processes
where appropriate and available. Such materials should be used to monitor nucleic acid extraction and
purification procedures. Internal controls should be considered to determine the nucleic acid quality of
each target sequence.
NOTE The CLSI MM17-A guideline provides recommendations for various aspects of verification and
[24]
validation of multiplex testing .
4.2 Multiplex molecular test quality nucleic acid and evaluation
4.2.1 Evaluation of nucleic acid quality for multiplex molecular tests
“PCR quality DNA” is described in ISO 22174, ISO 16577 and ISO 20395. In ISO 16577, it is described as a
DNA template of sufficient length, quality and structural integrity to be amplified by PCR, and “RT-PCR
quality RNA”, also described in ISO 22174, is an RNA template of sufficient length and quantity suitable
for reverse transcription and PCR. These cannot be applied to the various measurement methods of
multiplex molecular tests including PCR or RT-PCR-based methods, microarray, and massive parallel
sequencing.
Considering that multiplex molecular test is a molecular biological technique capable of simultaneous
detection of multiple nucleic acid sequence even of nucleic acids with shorter length, the evaluation
methods for multiplex molecular test quality nucleic acid suitable for each measuring system shall be
developed.
The nucleic acid quality should be determined prior to use in multiplex molecular tests. The method by
which nucleic acid quality is determined will depend on several factors such as the multiplex method
to be used, the known or anticipated quantity of nucleic acid present in the sample and the nucleic acid
to be analysed (DNA or RNA). The user shall choose the most appropriate approach, depending on the
subsequent multiplex molecular test to be used.
In order to determine quantity, concentration, purity and potential degradation of nucleic acid in
a sample, evaluation by spectrophotometric and/or fluorometric methods and/or gel or capillary
electrophoresis can be used. The nucleic acid quality should be estimated based on the size distribution
shown by electrophoresis of nucleic acids, the detection or quantity of representative genes (for
example, internal control genes including house-keeping genes). Spiked internal controls (i.e. samples
to which the analyte of interest has been added in a precisely known amount) can be used to determine
the nucleic acid quality of each target sequence to determine the process control. There are methods
available to evaluate the quality of nucleic acid present in a solution, as described in Annexes A to D.
EXAMPLE 1 The quality of RNA can be evaluated by electropherogram by means of the ratio of 28S:18S
ribosomal RNA of sample containing total RNA, the RNA integrity number (RIN value), or RNA integrity score
[31][32]
(RIS) .
While ribosomal RNA based quality metrics are popular for those conducting RNA analysis, they may
[33]
not always be useful for evaluating the quality of messenger RNA .
EXAMPLE 2 A260/A280 and A260/A230 ratios can be used to assess the purity of RNA
When multiplex molecular tests are designed to detect or quantify targets of different lengths it should
be ensured that the nucleic acid template is of sufficient quality as to be amplifiable across the target
size range. This can be achieved by use of a PCR reaction with primer sets that produce fragments
assessing the lower and upper target size range. Quality of the nucleic acid template suitable for the
assay can then be estimated based on the size distribution of the resultant PCR fragments using
capillary or gel electrophoresis.
For certain multiplex molecular tests, a critical aspect to ensuring quality of the nucleic acid sample
is to determine if the nucleic acid of interest is present. For example, the multiplex analysis of a low
abundance nucleic acid target from a specific source tissue or organism should ensure that the sample
contains nucleic acid from that tissue/organism. This confirmation may be a component included
within the multiplex test design or can be an external assay performed prior to the use of the nucleic
acid sample for multiplex assay.
NOTE To improve detection of viable pathogens, simultaneous measurement for ribosomal RNA or
messenger RNA can be used. Another approach could be considered to treat bacteria with DNA intercalators that
penetrate inactivated cells and inhibit PCR amplification but are excluded from viable cells.
The nucleic acid sample shall have a sufficient quantity of nucleic acid sequence of interest, which is
determined by the variant and population of interest. When multiplex molecular tests are designed
to detect or quantify different sequences of interest, sufficient amount required in a given subsequent
measurement method to fit for purpose shall be ensured. Measurement of the sequence of target with
lower prevalence relative to the others shall be ensured.
EXAMPLE For multiplex ligation-dependent probe amplification (MLPA), two denaturation fragments
(D-fragments) are used to indicate poor DNA denaturation due to salt contaminants in the DNA sample.
4.2.2 Evaluation of nucleic acid quantity
Appropriate methods for evaluating nucleic acid quantity shall be selected that are fit for purpose of the
test design of the multiplex molecular test.
Quantitation of the purified nucleic acid for analysis in a multiplex molecular test should measure
the quantity of the target genetic element, relative to the quantity of a specific reference, appropriate
calibrants and controls in order to compare the relative values of multiple variables. In this case, the
principle of quantitation is to determine the ratio (expressed as a percent) of two DNA or RNA target
sequences; i.e. a sequence of interest and a control gene or material (such as internal control genes,
reference materials). This confirmation may be a component included within the multiplex test design
or can be an external assay performed prior to the use of the nucleic acid sample for multiplex assay.
5 Procedure for preparation of nucleic acid
5.1 General
The nucleic acid extraction method employed shall be appropriate to obtain the quality and quantity of
nucleic acid required for the subsequent analysis.
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Evaluation of nucleic acids should be performed for their suitability in the multiplex molecular test
system in its final configuration, rather than suitability in separate singleplex reactions of which the
multiplex test is comprised.
5.2 Preparation of samples
5.2.1 General
In the pre-analytical phase, there are many stages of specimen handling including collection, fixation,
storage, transport, preparation, and processing. Since these pre-analytical factors greatly impact
the quality of a sample and subsequent test results, the appropriate handling of specimen shall be
[6][7][8][30]
ensured .
As the number of targets of interest increases in a multiplex assay, false negative results for certain
sequences can become more problematic. In particular, the quality as it pertains to the target with
the lowest abundance within the nucleic acid sample should be assessed, as competition for reaction
components can affect rare targets more significantly than more abundant targets.
EXAMPLE 1 In multiplex tests for detection of human papilloma virus subtypes in cervical infection, a poor-
quality specimen from uterine cervix, such as mucous without a sufficient number of human cells, can occur
during specimen collection. This can result in a false negative test due to an inadequate specimen. To validate
a negative test result in samples that are potentially sub-optimal, simultaneous detection of a human genome
sequence can be used as an internal assay control.
EXAMPLE 2 In multiplex tests for somatic variations in cancer, relative decrease in the targets of interests
can occur when non-neoplastic cells such as inflammatory infiltrates or endothelial cells for example are larger
in the proportion than the neoplastic cells, resulting in underestimates or false negative results for the target
sequence.
EXAMPLE 3 In multiplex tests for detection of microbial pathogens in respiratory tract infection, poor quality
nucleic acid due to a high viscosity of specimen can occur during collection and preparation of respiratory
specimens, resulting in false negative tests for a species with a low abundance. This can be minimized by
dissolving the specimen in NALC (N-acetyl-L-cysteine) and semi-alkali-proteases.
As the number of targets of interests increases, false positive results without clinical relevance becomes
more problematic. Thus, the clinical specimen should be collected and handled in a manner to avoid a
false positive result due to contamination of such an influence. When it cannot be avoided, the influence
should be assessed, with an appropriate method such as using the quantitative measurement with cut-
off values.
EXAMPLE 1 In multiplex tests for detection of bacterial pathogens in blood stream infection, contamination
can occur during blood collection, resulting in false positive tests for normal human skin flora such as coagulase-
negative staphylococcus. This can be reduced by aseptic techniques at blood collection, including antisepsis of
skin, hand hygiene and pre-packaged kits. Contamination of non-viable bacteria can also occur in blood after
antimicrobial treatment, resulting in positive tests for such bacteria. This can be reduced by avoiding blood
collection before or right after antimicrobial administration.
EXAMPLE 2 In multiplex tests for detection of mycobacterium species in respiratory tract infection,
contamination can occur during collection of respiratory specimens, resulting in positive tests for normal
environment, such as M. gordonae, M. chelonae or M. simiae. This can be reduced by use of a sterile collection
device such as bronchoscopy.
5.2.2 Consideration on tissue preparation
When methods based on enzymatic amplification reactions are used for multiplex molecular tests,
inhibitors present within tissue specimens may interfere with the detection or quantification of low-
level targets of interest relative to more abundant targets. Therefore, inhibitors shall be minimized as
much as possible, to an extent that ensures the measurement of each sequence of interest.
The presence and proportion of tumour cells should be evaluated to ensure interpretation of the test
result, and to determine whether the enrichment of tumour cells is needed to an extent that ensures the
measurement of each sequence
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