ISO/FDIS 4307

Molecular in vitro diagnostic examinations -- Specifications for pre-examination processes for saliva -- Isolated human DNA

Analyse de diagnostic moléculaire in vitro -- Spécifications relatives aux processus préanalytiques pour la salive -- ADN humain extrait

General Information

• Status: Published
• ICS: 11.100.10 - In vitro diagnostic test systems
• Technical Committee: ISO/TC 212 - Clinical laboratory testing and in vitro diagnostic test systems
• Drafting Committee: ISO/TC 212/WG 4 - Microbiology and molecular diagnostics
• Current Stage: 5020 - FDIS ballot initiated: 2 months. Proof sent to secretariat
• Start Date: 23-Jul-2021
• Completion Date: 23-Jul-2021

Standards Content (sample)

FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 4307
ISO/TC 212
Molecular in vitro diagnostic
Secretariat: ANSI
examinations — Specifications for
Voting begins on:
2021­07­23 pre-examination processes for saliva
— Isolated human DNA
Voting terminates on:
2021­09­17
Analyse de diagnostic moléculaire in vitro — Spécifications relatives
aux processus préanalytiques pour la salive — ADN humain extrait
ISO/CEN PARALLEL PROCESSING
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 4307:2021(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 2021
---------------------- Page: 1 ----------------------
ISO/FDIS 4307:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

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
CH­1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/FDIS 4307:2021(E)
Contents Page

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

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

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

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

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

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

5 Activities outside the laboratory ......................................................................................................................................................... 5

5.1 Specimen collection ............................................................................................................................................................................ 5

5.1.1 Information about the specimen donor/patient .................................................................................. 5

5.1.2 Selection of the saliva collection device by the laboratory .......................................................... 5

5.1.3 Saliva specimen collection from the donor/patient and stabilization procedures 6

5.1.4 Information on the specimen and storage requirements at saliva

collection facility/site .................................................................................................................................................. 7

5.2 Transport requirements ................................................................................................................................................................. 8

5.2.1 General...................................................................................................................................................................................... 8

5.2.2 Using saliva collection devices with DNA stabilizers ....................................................................... 8

5.2.3 Using saliva collection devices without DNA stabilizers ............................................................... 8

6 Activities inside the laboratory ............................................................................................................................................................. 9

6.1 Specimen reception ............................................................................................................................................................................ 9

6.2 Storage requirements ........................................................................................................................................................................ 9

6.3 Isolation of the saliva DNA ............................................................................................................................................................ 9

6.3.1 General...................................................................................................................................................................................... 9

6.3.2 Using commercial kit ........................................................................................................................................... ......10

6.3.3 Using the laboratory’s own protocol ...........................................................................................................10

6.4 Quantity and quality assessment of isolated DNA ................................................................................................10

6.5 Storage of isolated saliva DNA ................................................................................................................................................11

6.5.1 General...................................................................................................................................................................................11

6.5.2 Saliva DNA isolated with commercially available kits ..................................................................11

6.5.3 Saliva DNA isolated with the laboratory’s own protocols .........................................................11

Bibliography .............................................................................................................................................................................................................................12

© ISO 2021 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO/FDIS 4307:2021(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 non­governmental, 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, in collaboration with the European Committee for Standardization (CEN)

Technical Committee CEN/TC 140, in vitro diagnostic medical devices, in accordance with the Agreement

on technical cooperation between ISO and CEN (Vienna Agreement).

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 2021 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/FDIS 4307:2021(E)
Introduction

Molecular in vitro diagnostics has enabled a significant progress in medicine. Further progress is

expected by new technologies analysing profiles of nucleic acids, proteins, and metabolites in human

tissues and body fluids. However, the profiles of these molecules can change drastically during

specimen collection, transport, storage and processing thus making the outcome from diagnostics or

research unreliable or even impossible because the subsequent analytical assay will not determine the

situation in the patient but an artificial profile generated during the pre-examination process.

Genetic examination of DNA is commonly used in clinical practice. This includes e.g. predisposition

testing, pharmacogenomics, analysis of genetic disorders with the perspective used in precision

medicine. This is a fast-growing field in molecular diagnostics.

Saliva is increasingly used as a non-invasive alternative specimen to blood for the examination of

human DNA. Saliva naturally contains microorganisms and extraneous substances (e.g. food debris),

which make the composition of saliva more complex and unique among patients/donors. Dedicated

measures are therefore needed for informing and preparing patients/donors for the collection and

to check compliance with the instructions, in order to reduce the specimen variability. In contrast to

invasive specimen collection, saliva collection does not require trained and educated professionals or

dedicated facilities. By good instruction and verified collection device safety claims, saliva specimens

can be self-collected at home; however, home collection also contributes to high variability in specimen

quality. Similarly, medical laboratories/in vitro manufacturers need to be aware of specimen variability

when performing design verification and validation.

DNA in saliva can fragment or degrade after collection. In addition, bacteria present in the saliva

specimen can continue to grow, thus diluting the human DNA. DNases secreted by these bacteria can

also accelerate the DNA degradation. This can impact the sensitivity and reliability of DNA examination.

Standardization of the entire process from specimen collection to the DNA examination is needed to

minimize pre-examination impacts such as DNA degradation and fragmentation after saliva collection.

This document describes special measures which need to be taken to obtain good quality saliva

specimen/samples and isolated DNA therefrom for human DNA examination.
In this document, the following verbal forms are used:
— “shall” indicates a requirement;
— “should” indicates a recommendation;
— “may” indicates a permission;
— “can” indicates a possibility or a capability.
© ISO 2021 – All rights reserved v
---------------------- Page: 5 ----------------------
FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 4307:2021(E)
Molecular in vitro diagnostic examinations —
Specifications for pre-examination processes for saliva —
Isolated human DNA
1 Scope

This document specifies requirements and recommendations on the handling, storage, processing and

documentation of saliva specimens intended for human DNA examination during the pre-examination

phase before a molecular examination is performed.

This document is applicable to molecular in vitro diagnostic examination including laboratory

developed tests performed by medical laboratories. It can also be used by laboratory customers, in

vitro diagnostics developers and manufacturers, biobanks, institutions and commercial organizations

performing biomedical research, and regulatory authorities.

Dedicated measures that need to be taken for saliva collected on absorbing material or by mouth

washes are not described in this document. Neither are measures for preserving and handling of native

saliva cell­free DNA, pathogens, and other bacterial or whole microbiome DNA in saliva described.

NOTE International, national or regional regulations or requirements can also apply to specific topics

covered in this document.
2  Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 15189, Medical laboratories — Requirements for quality and competence
ISO 15190, Medical laboratories — Requirements for safety
3  Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 15189 and the following apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
ambient temperature
unregulated temperature of the surrounding air
[SOURCE: ISO 20184­1:2018, 3.2]
3.2
analyte
component represented in the name of a measurable quantity
[SOURCE: ISO 17511:2003, 3.2, modified — The examples were not taken over.]
© ISO 2021 – All rights reserved 1
---------------------- Page: 6 ----------------------
ISO/FDIS 4307:2021(E)
3.3
examination performance
analytical test performance
analytical performance

accuracy, precision, and sensitivity of a test to measure the analyte (3.2) of interest

Note 1 to entry: Other test performance characteristics such as robustness, repeatability can apply as well.

[SOURCE: ISO 20184­1:2018, 3.4]
3.4
DNA stabilizers

compounds, solutions or mixtures that are designed to minimize degradation and fragmentation of

DNA (3.6)

[SOURCE: ISO 20186-2:2019, 3.5, modified — The term “genomic DNA in blood” has been replaced with

“DNA”.]
3.5
closed system

non-modifiable system provided by the vendor including all necessary components for the analysis (i.e.

hardware, software, procedures and reagents)
[SOURCE: ISO 20186­2:2019, 3.6]
3.6
DNA
DNA

polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded (ssDNA)

form
[SOURCE: ISO 22174:2005, 3.1.2]
3.7
examination
analytical test

set of operations having the object of determining the value or characteristics of a property

Note 1 to entry: Processes that start with the isolated measurand and include all kinds of parameter testing or

chemical manipulation for quantitative or qualitative examination.

[SOURCE: ISO 15189:2012, 3.7, modified — The term and definition is used here without the original

notes.]
3.8
examination provider
analytical test provider
entity that provides the specific analytical test
3.9
interfering substance

endogenous or exogenous substance (e.g. stabilization solution) that can be present in specimens and

that can alter an examination result
[SOURCE: ISO 20184­1:2018, 3.12]
3.10
microorganism
entity of microscopic size, encompassing bacteria, fungi and protozoa

[SOURCE: ISO 11139:2018, 3.176, modified — The term “viruses” was deleted from the definition.]

2 © ISO 2021 – All rights reserved
---------------------- Page: 7 ----------------------
ISO/FDIS 4307:2021(E)
3.11
pre-examination process
pre-analytical phase
pre-analytical workflow

process that starts, 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) (3.12),

transportation to and within the analytical laboratory, isolation of analytes (3.2), and ends when the

analytical examination begins

Note 1 to entry: The pre-examination phase includes preparative processes that influence the outcome of the

intended examination.

[SOURCE: ISO 15189:2012, 3.15, modified — An additional term was added, and more detail was

included.]
3.12
primary sample
specimen

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

more quantities or properties assumed to apply for the whole

[SOURCE: ISO 15189:2012, 3.16, modified — The term and definition is used here without the original

notes.]
3.13
proficiency testing

evaluation of participant performance against pre-established criteria by means of interlaboratory

comparisons

[SOURCE: ISO/IEC 17043:2010, 3.7, modified — Term and definition are used here without the original

notes.]
3.14
room temperature
temperature in the range of 18 °C to 25 °C, for the purpose of this document
Note 1 to entry: Local or national regulations can have different definitions.
[SOURCE: ISO 20186­2:2019, 3.22]
3.15
saliva
whole saliva

bio-fluid of the mouth composed mainly of secretion originating from the three major salivary glands

(parotids, submandibular and sublingual glands) and from salivary glands present in the oral cavity

3.16
saliva collection device
tube or other container in which the saliva (3.15) specimen (3.12) is collected
3.17
sample
one or more parts taken from a primary sample (3.12)
[SOURCE: ISO 15189:2012, 3.24, modified — The examples were not taken over.]
3.18
stability

ability of a specimen (3.12)/sample (3.17) material, when stored under specified conditions, to maintain

a defined property value within specified limits for a specified period of time

Note 1 to entry: The measurand constituent for the purpose of this document is isolated DNA (3.6).

© ISO 2021 – All rights reserved 3
---------------------- Page: 8 ----------------------
ISO/FDIS 4307:2021(E)

[SOURCE: ISO Guide 30:2015, 2.1.15, modified — Note 1 was not taken over. The following words

were replaced: “characteristic” by “ability”; “reference material” by “sample material”; “specified” by

“defined”.]
3.19
storage

prolonged interruption of the pre-analytical workflow (3.11) of a sample (3.17) or analyte (3.2)

respectively, or of their derivatives, under appropriate conditions in order to preserve their properties

Note 1 to entry: Long-term storage typically occurs in laboratory archives or in biobanks.

[SOURCE: ISO 20184-1:2018, 3.22, modified — Example in the definition was deleted.]

3.20
validation

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

use or application have been fulfilled

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

[SOURCE: ISO 9000:2015, 3.8.13, modified — Note 1 and 3 were not taken over.]
3.21
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 and Note 2 were not taken over.]

3.22
workflow
structured series of activities necessary to complete a task
[SOURCE: ISO 20184-1:2018, 3.26, modified — The word “structured” was added]
4 General considerations

For general statements on medical laboratory quality management systems and in particular on

primary sample collection, reception and handling (including avoidance of cross contaminations) see

ISO 15189 or ISO/IEC 17020. The requirements on laboratory equipment, reagents, and consumables

according to ISO 15189 shall be followed; ISO 15189 and ISO/IEC 17020 can also apply. For general

considerations on specimen collection, transport, receipt, handling, and storage, see ISO/TS 20658. For

biobanking, ISO 20387 can also apply.

All steps of a diagnostic workflow can influence the final examination result. Thus, the entire workflow,

including specimen/sample storage and transport conditions, and their impact on the stability of

biomolecules intended to be examined shall be specified, verified and validated for its intended use.

This includes the development of in vitro diagnostic (IVD) medical devices. The stability of the human

DNA should be investigated throughout the complete pre-examination process development. The

verification of performance claims as well as the validation of the intended examination shall take into

account the variability of the saliva specimen's quality.
4 © ISO 2021 – All rights reserved
---------------------- Page: 9 ----------------------
ISO/FDIS 4307:2021(E)

During the design and development of a saliva DNA based examination, a risk assessment shall be

performed (see also ISO 14971). Mitigation measures for eliminating or reducing identified risks shall

be established where required for ensuring the performance of the examination. This shall include the

pre-examination workflow steps.

Before or during the design of an examination, it should be investigated and ensured that the human

DNA quality parameters such as minimum DNA amount, size and purity required for the examination

is/are not compromised in a manner impacting the examination performance.

Safety requirements on specimen collection, transport and handling shall be in accordance with

relevant ISO standards such as ISO 15189 and ISO 15190.

During the whole pre-examination process, precautions shall be taken to avoid cross contamination

between different specimens/samples, for example by using single-use material whenever feasible or

appropriate cleaning procedures between processing of different specimens/samples.

For all pre-examination steps, the examination manufacturer's instructions shall be followed, if

provided.

Where, for justified reasons (e.g. unmet patient needs), a commercial product is not used in accordance

with the manufacturer's instructions, responsibility for its verification, validation, use and performance

lies with the laboratory.

The manufacturer's material safety data sheet should be considered before first use of any potentially

hazardous material (e.g. chemicals in stabilizers).
5 Activities outside the laboratory
5.1 Specimen collection
5.1.1  Information about the specimen donor/patient

The documentation shall include the identity of the specimen donor/patient, which can be in the form

of a code.
The documentation should include, but is not limited to:

a) the relevant health status of the primary sample donor/patient [e.g. healthy, disease type,

concomitant disease, demographics (e.g. age and sex)];

b) the information about medical treatment and special treatment prior to saliva collection (e.g.

anaesthetics, medications);
c) the type and the purpose of the examination requested;
d) the appropriate consent from the specimen donor/patient. See also ISO 15189.
5.1.2  Selection of the saliva collection device by the laboratory

The Saliva DNA examination manufacturer instructions should contain specifications on the saliva

collection device(s) to be used. Where the examination manufacturer specifies usage of dedicated

saliva collection device(s), these shall be used.

Where the examination manufacturer does not provide such specifications (e.g. due to former less

stringent legal frameworks), the saliva collection device(s) shall be specified, verified, validated and

documented by the laboratory.

The quality and quantity of human DNA can be influenced by inadequate saliva collection procedures,

[6],[7]
inappropriate storage/shipping conditions as well as DNA isolation procedures .
© ISO 2021 – All rights reserved 5
---------------------- Page: 10 ----------------------
ISO/FDIS 4307:2021(E)

In order to prevent bacterial growth, human DNA degradation and fragmentation for a standardized

collection, saliva specimens should be collected in specifically developed saliva collection devices

containing appropriate stabilizers.

Alternatively, saliva can be collected in devices without stabilizers, such as cryo-vials or polypropylene

tubes, if other appropriate preservation methods according to 5.1.4.3 are going to be used. The use of

such tubes is not recommended for self-collection outside the laboratory.

5.1.3  Saliva specimen collection from the donor/patient and stabilization procedures

5.1.3.1 General
A protocol for the saliva specimen collection shall be in place.

For saliva collection a written and visual instruction, for example from the manufacturer, the laboratory

or physician, shall be supplied to the patient/donor.

The saliva DNA examination manufacturer instructions should contain specifications and instructions

on the saliva specimen collection procedure.

Where the examination manufacturer does not provide such specifications (e.g. due to former less

stringent legal frameworks), the saliva collection procedure shall be specified, verified and documented

by the laboratory.
The instruction for specimen collection shall include:

a) all the requirements necessary for the collection, identification, storage and transport to the

laboratory facility;

b) recommendations to follow before the collection, in particular related to nutrition, oral hygiene,

[8]
such as fasting before specimen collection ;

c) recommendation for collection to only submit saliva and avoid mucous from coughing/

expectoration or nasal secretions (sneezing).

For self­collection, the donor/patient shall be provided with an appropriate saliva collection device,

identity tags [e.g. label, Radio Frequency Identification (RFID)], and in general anything needed for the

specimen collection, storage and transport.

The donor/patient shall also be provided with an option to confirm conformance with the supplied

instruction for the saliva specimen collection, e.g. electronic, paper based.

The identity of the person collecting the specimen, which can be in the form of a code, and the time and

date of saliva collection according to ISO 15189 shall be documented.

For the labelling (sample/specimen identification) of the saliva collection tube a routine procedure

ISO 15189 or a procedure with additional information (e.g. 2D­barcode) shall be used.

Saliva collection devices shall be used in accordance with the supplied instructions, in particular:

1) to collect the required saliva volume;

2) to mix the saliva with the stabilizers immediately after saliva collection, e.g. by shaking or inverting.

NOTE Unless additives in the saliva collection device are homogenously mixed with the specimen, the saliva

DNA quality and quantity can be compromised, which can impact the validity and reliability of the examination

results.
Any tampering with and/or additions to the specimen shall be documented.

The donor/patient or the person collecting the saliva specimen from the donor/patient shall confirm

conformance with the supplied instruction for the saliva specimen collection.
6 © ISO 2021 – All rights reserved
---------------------- Page: 11 ----------------------
ISO/FDIS 4307:2021(E)
5.1.3.2 Using saliva collection devices with DNA stabilizers

The examination manufacturer should provide specified and verified instructions for saliva collection

(e.g. specific collection device, specimen mixing with stabilizer, etc.) which shall be followed.

Where the examination manufacturer does not provide such specifications (e.g. due to former less

stringent legal frameworks), the procedure shall be specified, verified and documented by the

laboratory. Instructions shall be written accordingly for the user and shall be followed. The saliva

collection device manufacturer specifications on storage and transport conditions can serve as a basis/

framework for the laboratory's own examination specific verification. These should include instructions

for preparation of the patient / donor, such as not to eat, drink, smoke, kiss or chew any substance for at

least 30 min before the saliva specimen collection.

NOTE Collection devices with stabilizers usually allow longer transport and storage durations prior to

testing.
5.1.3.3 Using saliva collection devices without DNA stabilizers

Where the examination manufacturer allows usage of saliva DNA collection devices without stabilizers,

they shall provide specified and verified instructions for the saliva collection which shall be followed.

Where the examination manufact
...