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

This document specifies requirements and recommendations for the pre-examination phase of human specimens, including saliva, skin, urine and stool, intended for microbiome DNA examination. The pre-examination phase includes but is not limited to specimen collection, handling, storage, processing and documentation.
This document is applicable to molecular in vitro diagnostic examinations 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.
Different dedicated measures are taken for the pre-examination phase for infectious disease examination (eg. targeted pathogen identification). These are not described in this document.
Different dedicated measures are taken for the pre-examination phase of saliva for human genomic DNA examination. These are not described in this document but are covered in CEN WI00140116, Molecular in vitro diagnostic examinations — Specifications for pre-examination processes for saliva — Isolated DNA.
NOTE International, national or regional regulations or requirements can also apply to specific topics covered in this document.

Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für präanalytische Prozesse für menschliche Proben - Isolierte Mikrobiom-DNA

Analyses moléculaires de diagnostic in vitro - Spécifications relatives aux processus préanalytiques pour les échantillons humains - ADN du microbiote isolé

Molekularne diagnostične preiskave in vitro - Specifikacije za predpreiskovalne procese za vzorce človeškega tkiva - Izolirana mikrobiom DNA

General Information

Status
Published
Public Enquiry End Date
09-Feb-2021
Publication Date
24-May-2021
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
13-May-2021
Due Date
18-Jul-2021
Completion Date
25-May-2021

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SLOVENSKI STANDARD
SIST-TS CEN/TS 17626:2021
01-julij-2021
Molekularne diagnostične preiskave in vitro - Specifikacije za predpreiskovalne
procese za vzorce človeškega tkiva - Izolirana mikrobiom DNA

Molecular in vitro diagnostic examinations - Specifications for pre-examination processes

for human specimen - Isolated microbiome DNA
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für menschliche Proben - Isolierte Mikrobiom-DNA

Analyses moléculaires de diagnostic in vitro - Spécifications relatives aux processus

préanalytiques pour les échantillons humains - ADN du microbiote isolé
Ta slovenski standard je istoveten z: CEN/TS 17626:2021
ICS:
11.100.10 Diagnostični preskusni In vitro diagnostic test
sistemi in vitro systems
SIST-TS CEN/TS 17626:2021 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST-TS CEN/TS 17626:2021
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SIST-TS CEN/TS 17626:2021
CEN/TS 17626
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
May 2021
TECHNISCHE SPEZIFIKATION
ICS 11.100.01
English Version
Molecular in vitro diagnostic examinations - Specifications
for pre-examination processes for human specimen -
Isolated microbiome DNA

Analyses moléculaires de diagnostic in vitro - Molekularanalytische in-vitro-diagnostische Verfahren

Spécifications relatives aux processus préanalytiques - Spezifikationen für präanalytische Prozesse für

pour les échantillons humains - ADN du microbiote menschliche Proben - Isolierte Mikrobiom-DNA

isolé

This Technical Specification (CEN/TS) was approved by CEN on 19 March 2021 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to

submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS

available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in

parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 17626:2021 E

worldwide for CEN national Members.
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SIST-TS CEN/TS 17626:2021
CEN/TS 17626:2021 (E)
Contents Page

European foreword ....................................................................................................................................................... 4

Introduction .................................................................................................................................................................... 5

1 Scope .................................................................................................................................................................... 6

2 Normative references .................................................................................................................................... 6

3 Terms and definitions ................................................................................................................................... 6

4 General considerations .............................................................................................................................. 11

5 Outside the laboratory ............................................................................................................................... 12

5.1 Specimen collection ..................................................................................................................................... 12

5.1.1 General ............................................................................................................................................................. 12

5.1.2 Information about the patient/specimen donor .............................................................................. 13

5.1.3 Specific information about the patient/specimen donor .............................................................. 13

5.1.4 Selection of specimen collection method and device(s) ................................................................ 15

5.1.5 Specimen collection from the patient/specimen donor and specimen stabilization.......... 16

5.2 Specimen storage and transport ............................................................................................................ 18

5.2.1 General ............................................................................................................................................................. 18

5.2.2 Using collection devices with stabilizers ............................................................................................. 19

5.2.3 Using collection devices without stabilizers ...................................................................................... 19

6 Inside the laboratory .................................................................................................................................. 20

6.1 Specimen reception ..................................................................................................................................... 20

6.2 Processing of specimens ............................................................................................................................ 20

6.3 Specimen storage before microbiome DNA isolation ..................................................................... 21

6.4 Isolation of microbiome DNA ................................................................................................................... 21

6.4.1 General ............................................................................................................................................................. 21

6.4.2 Using a commercial kit ............................................................................................................................... 22

6.4.3 Using a laboratory developed procedure ............................................................................................ 23

6.5 Quantity and quality assessment of isolated microbiome DNA .................................................. 23

6.5.1 General ............................................................................................................................................................. 23

6.5.2 Quantity assessment ................................................................................................................................... 24

6.5.3 Quality assessment ...................................................................................................................................... 24

6.6 Storage of isolated microbiome DNA .................................................................................................... 24

6.6.1 General ............................................................................................................................................................. 24

6.6.2 Microbiome DNA isolated using a commercial kit ........................................................................... 25

6.6.3 Microbiome DNA isolated using a laboratory developed procedure ........................................ 25

Annex A (informative) Impact of various pre-analytical variables on microbiome DNA

quantity, quality and profile .................................................................................................................... 26

A.1 Introduction ................................................................................................................................................... 26

A.2 Result – Impact of specimen stabilization method on isolated microbiome DNA

quantity ............................................................................................................................................................ 26

A.2.1 General ............................................................................................................................................................. 26

A.2.2 Method ............................................................................................................................................................. 26

A.2.3 Result/conclusion ........................................................................................................................................ 27

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CEN/TS 17626:2021 (E)

A.3 Result – Impact of specimen to stabilizer mass/volume ratio on isolated microbiome

DNA quantity and quality........................................................................................................................... 28

A.3.1 General ............................................................................................................................................................. 28

A.3.2 Method .............................................................................................................................................................. 28

A.3.3 Result/conclusion ........................................................................................................................................ 28

A.4 Result – Impact of different microbiome DNA isolation methods on microbiome DNA

profile................................................................................................................................................................ 29

A.4.1 General ............................................................................................................................................................. 29

A.4.2 Method .............................................................................................................................................................. 29

A.4.3 Result/conclusion ........................................................................................................................................ 30

A.5 Results – Impact of stabilization status and storage of collected specimens/samples

on microbiome DNA profile ...................................................................................................................... 31

A.5.1 General ............................................................................................................................................................. 31

A.5.2 Method .............................................................................................................................................................. 31

A.5.3 Result/conclusion ........................................................................................................................................ 32

Annex B (informative) Importance of using an in-process quality control material .......................... 33

B.1 Introduction ................................................................................................................................................... 33

B.2 Results .............................................................................................................................................................. 33

B.2.1 General ............................................................................................................................................................. 33

B.2.2 Method .............................................................................................................................................................. 33

B.2.3 Result ................................................................................................................................................................ 35

B.3 Conclusions ..................................................................................................................................................... 35

Bibliography ................................................................................................................................................................. 36

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CEN/TS 17626:2021 (E)
European foreword

This document (CEN/TS 17626:2021) has been prepared by Technical Committee CEN/TC 140 “In vitro

diagnostic medical devices”, the secretariat of which is held by DIN.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

According to the CEN/CENELEC Internal Regulations, the national standards organisations of the

following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,

Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

United Kingdom.
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CEN/TS 17626:2021 (E)
Introduction

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

expected using new technologies analysing the microbiome (e.g. bacteria, fungi, viruses, yeasts,

archaea) in human specimens.

The human microbiome has come into focus in many medical disciplines such as gastroenterology,

dermatology, or gynaecology as a potential biomarker for diagnosis and management of diseases, and

even as a therapeutic agent. Technologies analysing microbiome DNA such as shotgun metagenome or

amplicon-based sequencing (e.g. 16S or 18S rRNA gene sequencing) have accelerated this process and

are being increasingly performed in research and clinical practice.

However, the human microbiome profile can change drastically during the pre-examination process,

which includes the specimen collection, transport, storage, and processing. These changes can, for

example, be due to contamination of specimens with microbial cells or DNA from other sources than the

sampling site or due to undesired growth and/or instability of individual microorganisms and viruses.

Consequently, this makes the outcome from diagnostics or research unreliable or even impossible

because the subsequent microbiome DNA examination might not determine the real situation in the

patient but an artificial profile generated during the pre-examination processes. Therefore, special

measures have to be taken to secure the stability of the microbiome profile.

Specimens for microbiome analysis are often collected by donors/patients. Therefore, dedicated

measures are needed for informing donors/patients about and preparing them for the collection,

storage and transport of specimens, and to check the compliance with the instructions, in order to

reduce specimen variability.

In addition, isolation of microbiome DNA, which is representative in composition of the in vivo

microbiome of the respective body site, is critical. This can be especially challenging e.g. due to different

lysis requirements of the microorganisms (e.g. Gram-negative versus Gram-positive bacteria, or versus

fungi) as well as inhibitory compounds (e.g. PCR inhibitors) in the specimen, which can impact the

examination if not removed during the DNA isolation. The presence of high amounts of human host

DNA, in addition to DNA introduced by reagents such as remnant plasmid DNA from generation of

recombinant enzymes and/or DNA isolation kits, can further impact the examination result.

Therefore, standardization of the entire pre-examination workflow from specimen collection to the

microbiome DNA examination is needed.

Studies have been undertaken to determine the important influencing factors. This document draws

upon such work to codify and standardize the steps for microbiome DNA examination in what is

referred to as the pre-examination phase.
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.
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CEN/TS 17626:2021 (E)
1 Scope

This document specifies requirements and gives recommendations for the pre-examination phase of

human specimens, such as stool, saliva, skin and urogenital specimens, intended for microbiome DNA

examination. The pre-examination phase includes but is not limited to specimen collection, handling,

transport, storage, processing, isolation of DNA, and documentation.

This document is applicable to molecular in vitro diagnostic examinations 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.

Different dedicated measures are taken for pre-examination processes for infectious disease

examination (e.g. targeted pathogen identification) and for microbiome DNA examination from tissue

(e.g. biopsies). These are outside of the scope of this document.

Different dedicated measures are taken for pre-examination processes for saliva for human genomic

DNA examination. These are not described in this document but are covered in CEN/TS 17305,

Molecular in vitro diagnostic examinations — Specifications for pre-examination processes for saliva —

Isolated DNA.

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.

EN ISO 15189, Medical laboratories — Requirements for quality and competence (ISO 15189)

ISO 15190, Medical laboratories — Requirements for safety

ISO/TS 20658, Medical laboratories — Requirements for collection, transport, receipt, and handling of

samples
3 Terms and definitions

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

ones 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 https://www.electropedia.org/
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CEN/TS 17626:2021 (E)
3.1
aliquot

portion of a larger amount of homogeneous material, assumed to be taken with negligible sampling

error
Note 1 to entry: The term is usually applied to fluids.

Note 2 to entry: The definition is derived from the Compendium of Chemical Terminology Gold Book.

International Union of Pure and Applied Chemistry. Version 2.3.3., 2014; the PAC, 1990,62,1193 (Nomenclature

for sampling in analytical chemistry (Recommendations 1990)) p. 1206; and the PAC, 1990,62,2167 (Glossary of

atmospheric chemistry terms (Recommendations 1990)) p. 2173
3.2
ambient temperature
unregulated temperature of the surrounding air
3.3
analyte
component represented in the name of a measurable quantity
[SOURCE: EN ISO 17511:2003]
3.4
deoxyribonucleic acid
DNA

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

form
[SOURCE: EN ISO 22174:2005, 3.1.2]
3.5
deoxyribonuclease
DNase
enzyme that catalyzes the degradation of DNA [3.4] into smaller components
3.6
deviation
departure from an approved instruction, procedure and/or method

[SOURCE: EN ISO 15378:2017, 3.7.5 modified — The words “approved (3.7.1) standard operating

procedure (SOP) (3.7.10)” have been replaced by “instruction, procedure and/or method”.]

3.7
diagnosis

identification of a health or disease state from its signs and/or symptoms, where the diagnostic process

can involve examinations [3.8] and tests for classification of an individual's condition into separate and

distinct categories or subclasses that allow medical decisions about treatment and prognosis to be

made
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SIST-TS CEN/TS 17626:2021
CEN/TS 17626:2021 (E)
3.8
examination
analytical test

set of operations with the objective of determining the value or characteristics of a property

Note 1 to entry: Processes (i.e. set of operations) that start with the isolated analyte [3.3] and include all kinds

of parameter testing or chemical manipulation for quantitative or qualitative examination.

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

original Notes.]
3.9
examination manufacturer
analytical test manufacturer
entity that manufactures and/or produces the specific analytical test [3.8]
3.10
examination performance
analytical test performance

accuracy, precision, and sensitivity of a test to measure the analyte [3.3] of interest

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

3.11
homogeneous
uniform in structure and composition
3.12
interfering substances

endogenous substances of a specimen [3.26]/sample [3.25] or exogenous substances (e.g. stabilization

reagent [3.28]) that can alter an examination result
3.13
laboratory developed procedure

modified commercially available in vitro diagnostic device or fully in house developed procedure

3.14
microbial biomass

measure of the mass (amount) of microbiome [3.15] in a specimen [3.26]/sample [3.25]

3.15
microbiome, human

entire community of all commensal, symbiotic and pathogenic microorganisms [3.18] and viruses inside

and on specific human body sites in a particular environment/habitat
[SOURCE: [1][2][3][4]]
3.16
microbiome DNA
microbial DNA

DNA [3.4] of the microorganisms [3.18] and DNA viruses comprising the human microbiome [3.15]

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CEN/TS 17626:2021 (E)
3.17
microbiome DNA profile
microbial DNA profile

amounts of DNA molecules from the microbiome [3.15] that are present in a specimen [3.26]/sample

[3.25] and can be measured in the absence of any losses, inhibition or interference

3.18
microorganisms

entity of microscopic size, encompassing bacteria, archaea, single celled eukaryotes (incl. fungi,

protozoa), and phages
[SOURCE: [1][2]]
3.19
nonconformity
non-fulfillment of a requirement
[SOURCE: EN ISO 9000:2015, 3.6.9, modified — Note 1 to entry deleted.]
3.20
pre-examination processes
pre-analytical workflow
pre-examination phase
pre-analytical phase

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 specimen(s) [3.24],

transportation to and within the medical laboratory, isolation of analytes [3.3], and ends when the

examination [3.8] begins

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

intended examination.

[SOURCE: EN ISO 15189:2012, 3.15, modified — An additional term has been added, the words

“primary sample(s)” have been replaced by “specimen(s)” and more details have been included.]

3.21
primary collection device

tool specifically intended by a manufacturer to obtain or obtain and contain or obtain, contain and

preserve a specimen [3.26] from the donor/patient

[SOURCE: EN ISO 18113-1:2009, 3.55, Modified – Notes to entry have been deleted, “apparatus” has

been changed to “tool, “to obtain or obtain and contain” has been added, “for in vitro diagnostic

examination” has been deleted.]
3.22
secondary collection device

container into which the specimen [3.26] is transferred from or together with the primary collection

device [3.21]
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CEN/TS 17626:2021 (E)
3.23
proficiency test

evaluation of participant performance against pre-established criteria by means of inter-laboratory

comparisons

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

notes to entry.]
3.24
room temperature
for the purposes of this document, temperature in the range of 18 °C to 25 °C
Note 1 to entry: Local or national regulations can have different definitions.
3.25
sample
one or more parts taken from a specimen [3.26]

[SOURCE: EN ISO 15189:2012, 3.24, modified — The words “primary sample(s)” have been replaced by

“specimen(s)” and the example has been omitted.]
3.26
specimen
primary sample

discrete portion of a body fluid, breath, hair, stool, or biological material mechanically taken off body or

organ surfaces (e.g. by swabs, brushes, tapes, spatulas or blades) or tissue taken for examination of one

or more quantities or properties assumed to apply for the whole
[SOURCE: EN ISO 15189:2012, 3.16, modified — Notes to entry have been omitted.]
3.27
stability

ability of a specimen [3.26]/sample [3.25] material, when stored under specified conditions, to

maintain a stated property value within specified limits for a specified period of time

Note 1 to entry: The analyte for the purpose of this document is DNA.

[SOURCE: ISO Guide 30:2015, 2.1.15, modified — The words “reference material” have been replaced by

“specimen/sample material”.]
3.28
stabilizers
stabilization reagents
microbiome DNA stabilizers

compounds, solutions or mixtures that are designed to minimize changes of the microbiome DNA

profile [3.17] in a specimen [3.26] or sample [3.25] (by inhibition of undesired growth or decline of

microorganisms [3.18] and viruses, and/or of degradation and fragmentation of DNA [3.4])

3.29
storage
maintenance of biological material under conditions appropriate for intended use
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CEN/TS 17626:2021 (E)
3.30
validation

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

use or application have been fulfilled

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

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

3.31
verification

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

Note 1 to entry: The term “verified” is used to designate the corresponding status.

Note 2 to entry: Confirmation can comprise activities such as:
— performing alternative calculations;

— comparing a new design specification with a similar proven design specification;

— undertaking tests and demonstrations; and
— reviewing documents prior to issue

[SOURCE: ISO 9000:2015, 3.8.12, modified — Note 1 and 2 to entry have been omitted. New Note 2 to

entry has been added.]
3.32
workflow
series of activities necessary to complete a task
[SOURCE: ISO 20166-1:2018, 3.30]
4 General considerations

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

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

EN ISO 15189, EN ISO/IEC 17025 or EN ISO/IEC 17020. The requirements on laboratory equipment,

reagents, and consumables according to EN ISO 15189 shall be followed; EN ISO/IEC 17025 and

EN ISO/IEC 17020 can also apply.

All steps of the pre-examination, examination and post-examination processes (i.e. the entire workflow)

can influence the diagnosis or research study results.

Thus, this entire workflow shall be specified, verified and validated during the development of the

examination, including in vitro diagnostic (IVD) medical devices. This includes specifically all pre-

examination process steps such as the examination request, preparation and identification of the

patient, collection of the primary sample(s), transportation to and within the medical laboratory,

storage and isolation of analytes. This shall also include determination of and information on the

stability of the specimen within the timeframe between taking the specimen and its analysis and

storage conditions such as duration, temperature limits and freeze/thaw cycles.

The microbiome profile can change drastically during the pre-examination phase [5][6][27][28].

Microbiome composition and densities are influenced by lifestyle conditions
...

SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17626:2021
01-februar-2021
Molekularne diagnostične preiskave in vitro - Specifikacije za predpreiskovalne
procese za vzorce človeškega tkiva - Izolirana mikrobiom-DNA
Molecular in vitro diagnostic examinations — Specifications for pre-examination
processes for human specimen — Isolated microbiome DNA
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für menschliche Proben - Isolierte Mikrobiom-DNA

Analyses moléculaires de diagnostic in vitro - Spécifications relatives aux processus

préanalytiques pour les échantillons humains - ADN du microbiote isolé
Ta slovenski standard je istoveten z: FprCEN/TS 17626
ICS:
11.100.10 Diagnostični preskusni In vitro diagnostic test
sistemi in vitro systems
kSIST-TS FprCEN/TS 17626:2021 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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kSIST-TS FprCEN/TS 17626:2021
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kSIST-TS FprCEN/TS 17626:2021
FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17626
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
November 2020
ICS 11.100.01
English Version
Molecular in vitro diagnostic examinations - Specifications
for pre-examination processes for human specimen -
Isolated microbiome DNA

Analyses moléculaires de diagnostic in vitro - Molekularanalytische in-vitro-diagnostische Verfahren

Spécifications relatives aux processus préanalytiques - Spezifikationen für präanalytische Prozesse für

pour les échantillons humains - ADN du microbiote menschliche Proben - Isolierte Mikrobiom-DNA

isolé

This draft Technical Specification is submitted to CEN members for Vote. It has been drawn up by the Technical Committee

CEN/TC 140.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.

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 supporting documentation.

Warning : This document is not a Technical Specification. It is distributed for review and comments. It is subject to change

without notice and shall not be referred to as a Technical Specification.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprCEN/TS 17626:2020 E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
kSIST-TS FprCEN/TS 17626:2021
FprCEN/TS 17626:2020 (E)
Contents Page

European foreword ....................................................................................................................................................... 4

Introduction .................................................................................................................................................................... 5

1 Scope .................................................................................................................................................................... 6

2 Normative references .................................................................................................................................... 6

3 Terms and definitions ................................................................................................................................... 6

4 General considerations .............................................................................................................................. 11

5 Outside the laboratory ............................................................................................................................... 12

5.1 Specimen collection ..................................................................................................................................... 12

5.1.1 General ............................................................................................................................................................. 12

5.1.2 Information about the patient/specimen donor .............................................................................. 13

5.1.3 Specific information about the patient/specimen donor for specimens from gastro-

intestinal tract ............................................................................................................................................... 13

5.1.4 Selection of specimen collection method and device(s) ................................................................ 14

5.1.5 Specimen collection from the patient/specimen donor and specimen stabilization.......... 16

5.2 Specimen storage and transport ............................................................................................................ 18

5.2.1 General ............................................................................................................................................................. 18

5.2.2 Using collection devices with stabilizers ............................................................................................. 18

5.2.3 Using collection devices without stabilizers ...................................................................................... 19

6 Inside the laboratory .................................................................................................................................. 20

6.1 Specimen reception ..................................................................................................................................... 20

6.2 Processing of specimens ............................................................................................................................ 20

6.3 Specimen storage before microbiome DNA isolation ..................................................................... 21

6.4 Isolation of microbiome DNA ................................................................................................................... 21

6.4.1 General ............................................................................................................................................................. 21

6.4.2 Using a commercial kit ............................................................................................................................... 22

6.4.3 Using a laboratory developed procedure ............................................................................................ 22

6.5 Quantity and quality assessment of isolated microbiome DNA .................................................. 23

6.5.1 General ............................................................................................................................................................. 23

6.5.2 Quantity assessment ................................................................................................................................... 23

6.5.3 Quality assessment ...................................................................................................................................... 23

6.6 Storage of isolated microbiome DNA .................................................................................................... 24

6.6.1 General ............................................................................................................................................................. 24

6.6.2 Microbiome DNA isolated using a commercial kit ........................................................................... 24

6.6.3 Microbiome DNA isolated using a laboratory developed procedure ........................................ 24

Annex A (informative) Impact of various pre-analytical variables on microbiome DNA

quantity, quality and profile .................................................................................................................... 25

A.1 Introduction ................................................................................................................................................... 25

A.2 Result - Impact of specimen stabilization method on isolated microbiome DNA

quantity ............................................................................................................................................................ 25

A.2.1 General ............................................................................................................................................................. 25

A.2.2 Method ............................................................................................................................................................. 25

A.2.3 Result/conclusion ........................................................................................................................................ 26

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A.3 Result – Impact of specimen to stabilizer mass/volume ratio on isolated microbiome

DNA quantity and quality........................................................................................................................... 27

A.3.1 General ............................................................................................................................................................. 27

A.3.2 Method .............................................................................................................................................................. 27

A.3.3 Result/conclusion ........................................................................................................................................ 27

A.4 Result – Impact of different microbiome DNA isolation methods on microbiome DNA

profile................................................................................................................................................................ 28

A.4.1 General ............................................................................................................................................................. 28

A.4.2 Method .............................................................................................................................................................. 28

A.4.3 Result/conclusion ........................................................................................................................................ 29

A.5 Results - Impact of stabilization status and storage of collected specimens/samples

on microbiome DNA profile ...................................................................................................................... 30

A.5.1 General ............................................................................................................................................................. 30

A.5.2 Method .............................................................................................................................................................. 30

A.5.3 Result/conclusion ........................................................................................................................................ 31

Annex B (informative) Importance of using an in-process quality control material ....................... 32

B.1 Introduction ................................................................................................................................................... 32

B.2 Results .............................................................................................................................................................. 32

B.2.1 General ............................................................................................................................................................. 32

B.2.2 Method .............................................................................................................................................................. 32

B.2.3 Result ................................................................................................................................................................ 34

B.3 Conclusions ..................................................................................................................................................... 34

Bibliography ................................................................................................................................................................. 35

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European foreword

This document (FprCEN/TS 17626:2020) has been prepared by Technical Committee CEN/TC 140 “In

vitro diagnostic medical devices”, the secretariat of which is held by DIN.
This document is currently submitted to the Vote on TS.
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Introduction

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

expected using new technologies analysing the microbiome (e.g. bacteria, fungi, viruses, yeasts,

archaea) in human specimens.

The human microbiome has come into focus in many medical disciplines such as gastroenterology,

dermatology, or gynaecology as a potential biomarker for diagnosis and management of diseases, and

even as a therapeutic agent. Technologies analysing microbiome DNA such as shotgun metagenome or

amplicon-based sequencing (e.g. 16S or 18S rRNA gene sequencing) have accelerated this process and

are being increasingly performed in research and clinical practice.

However, the human microbiome profile can change drastically during the pre-examination process,

which includes the specimen collection, transport, storage, and processing. These changes can, for

example, be due to contamination of specimens with microbial cells or microbiome DNA from other

sources than the sampling site or due to undesired growth and/or stability of individual

microorganisms and viruses. Consequently, this makes the outcome from diagnostics or research

unreliable or even impossible because the subsequent examination might not determine the real

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

Therefore, special measures have to be taken to secure the stability of the microbiome profile.

Specimens for microbiome analysis are often collected by donors/patients. Therefore, dedicated

measures are needed for informing donors/patients about and preparing them for the collection,

storage and transport of specimens, and to check the compliance with the instructions, in order to

reduce specimen variability.

In addition, isolation of microbiome DNA, which is representative in composition of the in vivo

microbiome of the respective body site, is critical. This can be especially challenging e.g. due to different

lysis requirements of the microorganisms (e.g. Gram-negative versus Gram-positive bacteria, or versus

fungi) as well as inhibitory compounds (e.g. PCR inhibitors) in the specimen, which can impact the

examination if not removed during the DNA isolation. The presence of high amounts of human host

DNA, in addition to DNA introduced by reagents such as remnant plasmid DNA from generation of

recombinant enzymes and/or DNA isolation kits, can further impact the examination result.

Therefore, standardization of the entire pre-examination workflow from specimen collection to the

microbiome DNA examination is needed.

Studies have been undertaken to determine the important influencing factors. This document draws

upon such work to codify and standardize the steps for microbiome DNA examination in what is

referred to as the pre-examination phase.
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.
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1 Scope

This document gives requirements and recommendations for the pre-examination phase of human

specimens, such as stool, saliva, skin and urogenital specimens, intended for microbiome DNA

examination. The pre-examination phase includes but is not limited to specimen collection, handling,

transport, storage, processing, isolation of DNA, and documentation.

This document is applicable to molecular in vitro diagnostic examinations 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.

Different dedicated measures are taken for pre-examination processes for infectious disease

examination (e.g. targeted pathogen identification) and for microbiome DNA examination from tissue

(e.g. biopsies). These are outside of the scope of this document.

Different dedicated measures are taken for pre-examination processes for saliva for human genomic

DNA examination. These are not described in this document but are covered in CEN/TS 17305,

Molecular in vitro diagnostic examinations — Specifications for pre-examination processes for saliva —

Isolated DNA.

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.

EN ISO 15189, Medical laboratories - Requirements for quality and competence (ISO 15189:2012,

Corrected version 2014-08-15)
EN ISO 15190, Medical laboratories — Requirements for safety

ISO/TS 20658, Medical laboratories — Requirements for collection, transport, receipt, and handling of

samples
3 Terms and definitions

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

ones 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
aliquot

portion of a larger amount of homogeneous material, assumed to be taken with negligible sampling

error
Note 1 to entry: The term is usually applied to fluids
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Note 2 to entry: The definition is derived from the Compendium of Chemical Terminology Gold Book.

International Union of Pure and Applied Chemistry. Version 2.3.3., 2014; the PAC, 1990,62,1193 (Nomenclature

for sampling in analytical chemistry (Recommendations 1990)) p. 1206; and the PAC, 1990,62,2167 (Glossary of

atmospheric chemistry terms (Recommendations 1990)) p. 2173
3.2
ambient temperature
unregulated temperature of the surrounding air
3.3
analyte
component represented in the name of a measurable quantity
[SOURCE: EN ISO 17511:2003]
3.4
analytical test performance

accuracy, precision, and sensitivity of a test to measure the analyte [3.3] of interest

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

well.
3.5
deoxyribonucleic acid
DNA

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

form
[SOURCE: EN ISO 22174:2005, 3.1.2]
3.6
deoxyribonuclease
DNase
enzyme that catalyzes the degradation of DNA [3.5] into smaller components
3.7
deviation
departure from an approved instruction, procedure and/or method

[SOURCE: EN ISO 15378:2017, 3.7.5 modified — The words “approved (3.7.1) standard operating

procedure (SOP) (3.7.10)” were replaced by “instruction, procedure and/or method”.]

3.8
diagnosis

identification of a health or disease state from its signs and/or symptoms, where the diagnostic process

can involve examinations [3.9] and tests for classification of an individual's condition into separate and

distinct categories or subclasses that allow medical decisions about treatment and prognosis to be

made
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3.9
examination
analytical test

set of operations with the objective of determining the value or characteristics of a property

Note 1 to entry: Processes (i.e. set of operations) that start with the isolated analyte [3.3] and include all kinds

of parameter testing or chemical manipulation for quantitative or qualitative examination.

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

original notes.]
3.10
examination manufacturer
analytical test manufacturer
entity that manufactures and/or produces the specific analytical test [3.9]
3.11
homogeneous
uniform in structure and composition
3.12
interfering substances

endogenous substances of a specimen [3.26]/sample [3.25] or exogenous substances (e.g. stabilization

reagent [3.28]) that can alter an examination result
3.13
laboratory developed procedure

modified commercially available in vitro diagnostic device or fully in house developed procedure

3.14
microbial biomass

measure of the mass (amount) of microbiome [3.15] in a specimen [3.26]/sample [3.25]

3.15
microbiome, human

entire community of all commensal, symbiotic and pathogenic microorganisms [3.18] and viruses inside

and on specific human body sites in a particular environment/habitat
[SOURCE: [1][2][3][4]]
3.16
microbiome DNA
microbial DNA

DNA [3.5] of the microorganisms [3.18] and DNA viruses comprising the human microbiome [3.15]

3.17
microbiome DNA profile
microbial DNA profile

amounts of DNA molecules from the microbiome [3.15] that are present in a specimen [3.26]/sample

[3.25] and can be measured in the absence of any losses, inhibition or interference

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3.18
microorganisms

entity of microscopic size, encompassing bacteria, archaea, single celled eukaryotes (incl. fungi,

protozoa), and phages
[SOURCE: [1][2]]
3.19
nonconformity
non-fulfillment of a requirement
[SOURCE: EN ISO 9000:2015, 3.6.9, modified — Note 1 to entry deleted.]
3.20
pre-examination processes
pre-analytical workflow
pre-examination phase
pre-analytical phase

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 specimen(s) [3.24],

transportation to and within the medical laboratory, isolation of analytes [3.3], and ends when the

examination [3.9] begins

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

intended examination.

[SOURCE: EN ISO 15189:2012, 3.15, modified — An additional term has been added, the words

“primary sample(s)” were replaced by “specimen(s)” and more details have been included.]

3.21
primary collection device

tool specifically intended by a manufacturer to obtain or obtain and contain or obtain, contain and

preserve a specimen [3.26] from the donor/patient

[SOURCE: EN ISO 18113-1:2009, 3.55, Modified – Notes to entry have been deleted, “apparatus” has

been changed to “tool, “to obtain or obtain and contain” has been added, “for in vitro diagnostic

examination” has been deleted.]
3.22
secondary collection device

container into which the specimen [3.26] is transferred from or together with the primary collection

device [3.21]
3.23
proficiency test

evaluation of participant performance against pre-established criteria by means of inter-laboratory

comparisons

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

notes to entry.]
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3.24
room temperature
for the purposes of this document, temperature in the range of 18 °C to 25 °C
Note 1 to entry: Local or national regulations can have different definitions.
3.25
sample
one or more parts taken from a specimen [3.26]

[SOURCE: EN ISO 15189:2012, 3.24, modified — The words “primary sample(s)” were replaced by

“specimen(s)” and the example has been omitted.]
3.26
specimen
primary sample

discrete portion of a body fluid, breath, hair, stool, or biological material mechanically taken off body or

organ surfaces (e.g. by swabs, brushes, tapes, spatulas or blades) or tissue taken for examination of one

or more quantities or properties assumed to apply for the whole
[SOURCE: EN ISO 15189:2012, 3.16, modified — NOTEs to entry have been omitted.]
3.27
stability

ability of a specimen [3.26]/sample [3.25] material, when stored under specified conditions, to

maintain a stated property value within specified limits for a specified period of time

Note 1 to entry: The analyte for the purpose of this document is DNA.

[SOURCE: ISO Guide 30:2015, 2.1.15, modified — The words “reference material” were replaced by

“specimen/sample material”.]
3.28
stabilizers
stabilization reagents
microbiome DNA stabilizers

compounds, solutions or mixtures that are designed to minimize changes of the microbiome DNA

profile [3.17] in a specimen [3.26] or sample [3.25] (by inhibition of undesired growth or decline of

microorganisms [3.18] and viruses, and/or of degradation and fragmentation of DNA [3.5])

3.29
storage
maintenance of biological material under conditions appropriate for intended use
3.30
validation

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

intended use or application have been fulfilled

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

[SOURCE: ISO 9000:2015, 3.8.13, modified — NOTE 1 and 3 have been omitted.]
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3.31
verification

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

Note 1 to entry: The term “verified” is used to designate the corresponding status.

[SOURCE: ISO 9000:2015, 3.8.12, modified — NOTE 1 and NOTE 2 have been omitted.]
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
3.32
workflow
series of activities necessary to complete a task
4 General considerations

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

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

EN ISO 15189, EN ISO/IEC 17025 or EN ISO/IEC 17020. The requirements on laboratory equipment,

reagents, and consumables according to EN ISO 15189 shall be followed; EN ISO/IEC 17025 and

ISO/IEC 17020 can also apply.

All steps of the pre-examination, examination and post-examination processes (i.e. the entire workflow)

can influence the diagnosis or research study results.

Thus, this entire workflow shall be specified, verified and validated during the development of the

examination, including in vitro diagnostic (IVD) medical devices. This includes specifically all pre-

examination process steps such as the examination request, preparation and identification of the

patient, collection of the primary sample(s), transportation to and within the medical laboratory,

storage and isolation of analytes. This shall also include determination of and information on the

stability of the specimen within the timeframe between taking the specimen and its analysis and

storage conditions such as duration, temperature limits and freeze/thaw cycles.

The microbiome profile can change drastically during the pre-examination phase [5][6][27][28].

Microbiome composition and densities are influenced by lifestyle conditions [7], and treatment of the

sampling site prior to specimen collection, and strongly differ depending on the body site and disease

state. The microbiome also differs from individual to individual. Different collection methods can vary

in microbial biomass yield and extent of human DNA contamination [6][7][8]. Upon removal of

specimens from the body undesirable growth or decline of certain microorganisms/viruses and

degradation and fragmentation of DNA can occur without rapid specimen stabilization by microbiome

DNA stabilizers, freezing or immediate microbiome DNA isolation [5][6]. Also, different DNA isolation

methods, particularly the lysis step, can influence the DNA profile and quantity as different

microorganisms
...

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