ISO 15189 Application Guidance Document for
Molecular-Genetic Examination

Japanese Committee for Clinical Laboratory Standards

ISO 15189 Application Guidance Document for
Molecular-Genetic Examination

Publisher
Editor-in-chief

Yasushi Takagi, MD, PhD
Hayato Miyachi, MD, PhD

Copyright© Japanese Committee for Clinical Laboratory Standards
Address for Editorial Correspondence:
Japanese Committee for Clinical Laboratory Standards, 2-7-13 Uchi-kanda, Chiyodaku, Tokyo 101-0047, Japan. URL：http://www.jccls.org/
Printed in Japan

2

Contents
Foreword
1.

Scope............................................................................................................. 6

2.

Normative references ＊. .................................................................................. ＊3.

Terms and definition. ........................................................................................ 7

4.

Management requirements................................................................................. 7

4.1

Organization and management responsibility......................................................... 7

4.2

Quality management system............................................................................... 8

4.3

Document control............................................................................................. 9

4.4

Service agreements ＊. .................................................................................... ＊4.5

Examination by referral laboratories................................................................... 10

4.6

External services and supplies ＊....................................................................... ＊4.7

Advisory services ＊......................................................................................... ＊4.8

Resolution of complaints.................................................................................. 11

4.9

Identification and control of nonconformities. ...................................................... 11

4.10 Corrective action............................................................................................ 13
4.11 Preventive action ＊......................................................................................... ＊4.12 Continual improvement ＊................................................................................. ＊4.13 Control of records........................................................................................... 14
4.14 Evaluation and audits...................................................................................... 15
4.15 Management review......................................................................................... ＊5.

Technical requirements................................................................................... 16

5.1

Personnel...................................................................................................... 16

5.2

Accommodation and environmental conditions.................................................... 22

5.3

Laboratory equipment, reagents, and consumables............................................... 24

5.4

Pre-examination processes. ............................................................................. 26

5.5

Examination processes.................................................................................... 31

5.6

Ensuring quality of examination results. ............................................................. 44

5.7

Post-examination processes............................................................................. 50

5.8

Reporting of results......................................................................................... 51

5.9

Release of results........................................................................................... 54

5.10 Laboratory information management. ................................................................. 55
6.

Reference. .................................................................................................... 60＊ ISO 15189 requirement itself has not been cited so that the original document should be referred.

3

■Foreword
To ensure the objectivity and reliability of medical laboratories performing molecular-genetic
examinations, it is necessary to meet the requirements of the Japanese version of “The Best Practice
Guidelines for Molecular-Genetic Examinations”. Medical laboratories should perform tests incompliance with ISO 15189 requirements, particularly when those are not covered by NHI (National
Health Insurance) such as laboratory-developed tests (LDTs).
In establishment of the accreditation program under ISO 15189, the outcome of activities related toquality assurance and standardization should be reflected to ensure the required level of the Japaneseversion of “Best Practice Guidelines for Molecular-Genetic Tests’.
Based on these, a research project of the Japan Agency for Medical Development (AMED) was
conducted to investigate fundamental structures to establish the ISO 15189 accreditation program formolecular-genetic examination as part of ISO/TC212 research project from FY 2017 to FY 2019.
The research subject was summarized as follows:1)

The development of a guidance document to clarify a basis of the criteria for accreditation;

2)

The development of proficiency tests for on-site evaluation;

3)

The development of external quality assessment program to monitor the performance of accredited
laboratories.

A basic policy for the development of the guidance document was assignment of relevant descriptionsof guidelines and academic literatures dedicated to molecular-genetic examinations to each of requirementsof ISO 15189. The references were added with their sources and put as notes, as necessary.
Regarding molecular-genetic examinations including NGS, the following recommendations andrequirements are described in this guidance document.
1)

personnel competences,2)

validation and verification of procedures,

3)

internal quality control methods,

4)

use of quality control materials,

5)external quality assessment program (such as an interlaboratory comparison program or proficiency
testing program),

6)

handling of patient specimens and confidentiality of patient genome information.

It would be useful to develop ISO 15189 application guidance on molecular-genetic tests for bothaccreditation bodies and medical laboratories.
Namely, the merits for the accreditation body include a guide for preparation of accreditation criteria,
the training of auditors and standardization of audit.
Meanwhile, the merits for medical laboratories include the implementation and improvement ofquality management system and technical management system.
4

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

As mentioned above, this guidance document would be useful for quality improvement of medicallaboratories performing molecular-genetic examinations.
Consequently, we sincerely hope that this guidance document will contribute to the promotion ofhigh-quality practice of genome-based medicine through appropriate medical laboratory service.
To use this guidance document as a supplement teaching aid in ISO 15189 Accreditation Support
Course of the Training Program, as part of AMED Asian Cancer Clinical Trials Network (ATLAS)
Project, the Japanese version was translated to English and edited in research project from FY 2020 to FY
2021.

Development of this guidance document was supported by the Promoting Platform of Genomics based
Drug Discovery (Project No. JP19kk0305005) and the Project Promoting Clinical Trials for Developmentof New Drugs and Medical Devices (Project No. JP21lk0201005j0001) from Japan Agency for Medical
Research and Development (AMED).

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

5

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

Scope

1

・ This guidance document specifies the quality and competence requirements in medical laboratories
that perform molecular and cytogenetic examinations that are used for medical care or whose
results are to be reported to patients.
A medical laboratory is a facility for the purpose of providing information on disease fordiagnosis, management, prevention, and treatment, or for health assessment. It performs biological,
microbiological, immunological, chemical, immunohematological, hematological, biophysical,
cytological, pathological, genetic, molecular, or other examinations using a biological sample
derived from the human body, and can provide consultation and advisory services covering all
aspects of medical laboratory work, including the interpretation of results and advice on further
appropriate examinations.
【Note】
・ Molecular/cytogenetic examination includes those approved and non-approved under the
Pharmaceutical and Medical Device Law. At present, research facilities are not covered in thescope of this guidance document in Japan. Internationally, research facilities are included in the
ISO 15189 accredited facilities.
・ Molecular examination is classified and defined as follows.
① Nucleic acid testing for pathogen: testing to detect and analyze the nucleic acid (DNA or RNA)of exogenous pathogens (viruses, bacteria, etc.) that cause infectious diseases in humans.
② Molecular-genetic testing for human somatic gene alterations: molecular examination to clarify
transient genetic information that can be changed with the disease state, confined to diseaselesions/tissues, such as to detect structural abnormalities of genes unique to cancer cells and gene
expression analysis.
③ Human genetic testing (molecular-genetic tests for human germline alterations): testing to reveal
the genetic information inherent in the individual (germline alterations information revealed bygenetic analysis) that does not change throughout the genome and mitochondria that theindividual naturally holds, such as monogenic diseases, multifactorial diseases, effects, side effects,
metabolism of drugs, or genetic testing related to individual identification.

2

Normative references＊

6

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

3

Terms and definition

3.7 Examination
・ The term “molecular-genetic examination” used herein refers to molecular examination andincludes the following three generic terms.
① Nucleic acid testing for pathogen: testing to detect and analyze nucleic acids (DNA or RNA) of
pathogens (microorganisms such as viruses and bacteria) that cause infectious diseases in humans② Molecular-genetic testing for human somatic gene alterations: testing that reveals transient genetic
information that is limited to diseased lesions/tissues, and can change with the symptoms, such asdetection of structural abnormalities of genes unique to cancer cells and gene expression profiling.
③ Human genetic testing (Molecular-genetic tests for human germline alterations): testing thatreveals genetic information inherent to the individual that does not change throughout the genome
and mitochondria that the individual naturally holds (germline alterations information revealed
by genetic analysis) such as monogenic diseases, multifactorial diseases, effects/adverse effects/
metabolism for drugs, and genetic testing related to individual identification.
④ Cytogenetic examination: “Molecular/cytogenetic examination” is generally referred to as“Molecular examination”. “Molecular examination” basically includes “Cytogenetic examination”
and the name is used only when the content is specialized for “Cytogenetic examination”.

4

Management Requirements

4.1 Organization and Management Responsibility
4.1.1.3 Ethical conducta)there is no involvement in any activities that would diminish confidence in the laboratory’s
competence, impartiality, judgement or operational integrity.
・ When conducting molecular examination, legal and ethical aspects should be taken into accountand performed in a scientifically and technically correct manner.

e)confidentiality of information is maintained.
・ In the case of adding detailed information on gene variants by receiving the gene sequence dataobtained from the molecular/cytogenetic examination performed at medical institutions orregistered clinical laboratories and analyzing the sequence data of the gene using dedicatedsoftware, programs, or databases, as it does not correspond to clinical laboratory tests as definedby the act on clinical laboratory technicians, it is not necessary to register as a registered clinicallaboratory.
・ However, the laboratory should not only inform relevant parties such as medical institutions orregistered clinical laboratories requesting analysis in advance about the conditions for submitting
gene sequence data and the reliability of additional information, but also refer to Registered

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

7

Clinical Laboratory Instruction Guidelines about preparation of standard operating procedures
and information security measures, if necessary.

4.1.2.7 Quality Manager
c)ensuring the promotion of awareness of users’ needs and requirements throughout the laboratoryorganization.

・ A person responsible for ensuring quality shall be appointed. It should be noted that the law thatpartially amends the Medical Care Act, etc., allows the concurrent appointment of a person in
charge of ensuring the quality of overall clinical laboratory tests. Regarding qualifications, expertisein all medical laboratory quality controls with clinical tests, including molecular examination,
appropriate experience, and quality, is required.
【Note】
Persons familiar with the quality control of both clinical and molecular examination are desirable.

4.2 Quality Management System
4.2.1 General Requirements【NGS Wet Bench Process—Quality Management Program】
・ The laboratory shall follow a documented quality management program for the NGS analytical
wet bench process.
・ All molecular examinations shall be carried out under a quality assurance framework, and thelaboratory shall be responsible for ensuring that the test results meet the purpose and that themethod used is appropriate by setting and maintaining the quality of the examination procedure.
Therefore, whether the quality assurance framework can be applied to laboratories that provide
molecular examination should be considered accordingly.
(Explanation)
・ The quality assurance framework for molecular examination includes all works that directly or
indirectly affect the quality of the examination work, such as training of technologists and experts,
promotion of quality assurance plans for standardization of protocols, acquisition of referencematerials, promotion of proficiency testing systems for testing laboratories, and provision of
information on the adaption and interpretation of molecular examination. In other words, notonly the technical aspects of the testing but also the assurance of safety, validity, and usefulness interms of medical and health care related to testing should be comprehensively considered.
【NGS bioinformatics pipeline quality management program】
・ The laboratory shall have a documented quality management program for the NGS bioinformatics
pipeline.
4.2.2 Documentation Requirements
8

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

4.2.2.1 Generale)copies of applicable regulations, standards and other normative documents.

・ As NGS-based clinical tests are emerging technologies and are much more complex than traditional
Sanger sequencing-based testing, new regulatory standards are being developed for laboratories
offering these tests.
4.3 Document Control
・ The laboratory shall create standard operating procedures (SOPs), strive to standardize operations,and perform all operations in accordance with the standardized SOPs. In addition, the laboratory
should maintain operation records and/or logs, history of non-conformities, errors, and corrections,and conduct continuous improvements. The laboratory shall create and operate appropriate reportformats for these documents. Additionally, they shall review them regularly and manage and store
them appropriately.
f)Changes to documents are identified.

【Edition Traceability (Version etc.)】
・ The specific version(s) of the bioinformatics pipeline used to generate NGS data files shall be
traceable for each patient report.
① The specific versions of each component and, where available, associated configurations (e.g.
command line parameters or other configuration items) of the bioinformatics pipeline used togenerate NGS data shall be traceable for each patient report. The bioinformatics pipeline foranalyzing NGS data, especially when based primarily on open-source software, is often composedof a combination of different software packages, scripts, and databases.
② The performance of a single software package or script and the composition of an internal orexternal database can significantly impact the overall performance of the bioinformatics pipeline.
As a result, it is important for the laboratory to be able to connect each patient report to theparticular bioinformatics pipeline used to generate the report.
③ For in-house scripts and software packages, changes in the script or software shall also be
documented, but documentation of each component of the pipeline does not need to appear in
the patient report. Rather, it is acceptable to refer to the entire pipeline using laboratory-specificdesignations (e.g. NGS pipeline v1.0.1).
④ Laboratory-specific designations are unique to a single combination of pipeline components
and configurations. Therefore, any change to a different version of a software package, script, orinternal or external database, or change to the configuration of any software, requires a new
unique laboratory-specific designation and assay re-validation.
j)

At least one copy of an obsolete controlled document is retained for a specified time period or inaccordance with applicable specified requirements.

Monitoring of Upgrades
ISO 15189 Application Guidance Document for Molecular-Genetic Examination

9

・ The laboratory shall have a policy for monitoring, implementing, and documenting upgrades to
instruments, sequencing chemistries, and reagents or kits used to generate NGS data.
① Laboratories should be aware of upgrades to ensure that they do not use obsolete methods.
② The laboratory shall implement a policy to monitor and implement upgrades to instruments,
sequencing chemistries, and reagents or kits used to generate NGS data. The policy should
address how laboratories performing NGS-based testing can ensure that they are using the most
up-to-date sample library preparation as appropriate for that assay, clone fragment amplification,and sequencing methods in this rapidly evolving environment, provided that these newer
methods have been validated by the laboratory to improve the quality, reproducibility, andaccuracy of the assay.
・ The policy should also address the methods used to monitor upgrades, and when a relevant
upgrade(s) will be implemented and validated before productive clinical use. For example, thelaboratory’s policy can be to monitor and implement upgrades at specified intervals (e.g. quarterly,
semiannual, or annually) depending on the relevance of the new upgrade for enhancing assay
performance. Additionally, since the implementation of upgrades can require revalidation of theentire wet bench process or at least the relevant steps, it can be convenient to set time intervals
accordingly.
4.4 Service agreements＊
4.5 Examination by Referral Laboratories
4.5.1 Selecting and Evaluating Referral Laboratories and Consultants
NGS testing referral policy
・ The laboratory shall have a policy for the selection of referral laboratories and other service
providers for NGS testing. Referral can include the entire NGS testing process or only the wetbench or bioinformatics process.
① The laboratory director or the designee is responsible for the selection of an external referrallaboratory or other service provider.
② The laboratory director or the designer ensures the quality of performance of the external
provider for NGS wet bench and/or bioinformatics services.
③ Some of the specific aspects that the laboratory director needs to consider when selecting referrallaboratories or service providers are ensuring that
(1) Turnaround times are acceptable for the clinical needs for which testing is being performed.
(2) The referral laboratory providing the analytic wet bench information (i.e., sequence generation)
meets the selection criteria as per CAP requirements.
(3) The quality of the results from the external bioinformatics service provider is verified to be10

ISO 15189 Application Guidance Document for Molecular-Genetic Examinationaccurate at a high level.
④ For evidence of compliance, copies of valid certificates are required for those who outsource thewet bench sequencing workflow.
⑤ Copies of in-house validation of laboratories providing bioinformatics analysis are required forlaboratories that outsource the bioinformatics workflow.
b)

Arrangements with referral laboratories and consultants are reviewed and evaluated periodically toensure that the relevant parts of this International Standard are met.

・ When a part of the process is referred, the laboratory accredited by ISO 15189 is responsible for
not only carrying out the process before and after the process (preparation of specimen,transportation, release of result report, etc.), but also for selecting and evaluating the referrallaboratories.
・ To satisfy international standards for molecular and cytogenetic examination, the laboratory
should maintain the system necessary for obtaining third-party accreditation (e.g. ISO 15189) byimplementing documentation.
4.6 External services and supplies＊
4.7 Advisory services＊
4.8 Resolution of Complaints
・ In branch laboratories and registered clinical laboratories, it is necessary to clarify the procedures
for resolution of complaints received from medical institutions that ordered the test, the recording
method, and the reporting method to medical institutions and governments.
Document name: Standard Operating Procedure for Resolution of Complaint
Major matters to be included.
① Complaint resolution system (including the role of the supervising doctor)② Complaint resolution procedure③ Matters concerning reporting to the medical institution that ordered the test and the government④ How to complete the complaint resolution log
4.9 Identification and Control of Nonconformitiesh)each episode of nonconformity is documented and recorded, with these records being reviewed atregular specified intervals to detect trends and initiate corrective action.

Exception Log
The laboratory maintains an exception log for patient samples (specimens), where steps used in
the NGS analytic wet bench process deviate from standard operating procedures (SOPs).
ISO 15189 Application Guidance Document for Molecular-Genetic Examination11

① The laboratory shall document any deviation from the SOP along with an explanation of thedeviation and the resulting outcome. Examples of anticipated deviations can include alteredprocessing upon receipt of a suboptimal specimen, changes to library preparation, and sequencingof libraries with suboptimal concentrations.
② Exceptions can pertain to the specimen quality and the examination process. At the time of
specimen accessioning, an assessment is made as to whether or not a sample is in optimal
condition for testing. If there is a concern, this shall be documented on the worksheet or on apending log and communicated to a supervisor or laboratory director.
③ The laboratory director can decide to proceed with the testing, but should communicate theissue to the ordering physician and document this communication electronically or on theworksheet.
One example of such a scenario is a specimen that was not transported under the optimal
conditions.
A decision can be made to process the sample and proceed with subsequent testing only if the
DNA sample is found to be adequate. (See Section 4.4.1 e).)④ Issues related to specific steps of the wet bench procedure shall be reported to the laboratory
supervisor or director. This allows the evaluation of whether the testing itself has been
compromised and whether the test can be completed.
⑤ If, after troubleshooting, the testing is evaluated as satisfactory, and the results can be interpretedby the laboratory director, provided that the quality controls of the test and sample results weredeemed adequate, all aspects of the testing issue(s) shall be thoroughly documented in an‘‘exception log,’’ including the troubleshooting, resolution, and pertinent communications(especially regarding who was involved and who was informed by whom and on what date), andcan be also incorporated into the monthly quality assurance report.
⑥ On occasion, the laboratory SOP itself may have to be revised to improve phrasing, makeprocess steps clearer, or remove small inaccuracies in order to optimize the protocol. In suchcases, the proposed correction should ideally be supported by at least two additional individuals,
including the laboratory supervisor and either the technologist who developed the assay or areference technologist. Any such corrections shall be approved, signed, and dated by the directorof the laboratory. This is not an exception log issue, but rather a correction in the manner inwhich the assay is described. (See Section 4.3 (i).)
Exception Log-Bioinformatics pipeline
・ The laboratory shall maintain an exception log for patient cases in which the examination steps
used in the bioinformatics pipeline deviate from standard operating procedures (SOPs).
① Deviations from the laboratory SOP during any examination step used in the bioinformatics
pipeline shall be documented in an exception log file, including any alterations in software
packages, script, version number, database, command line, or parameters.
12

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

③ Any failures arising during the bioinformatics process shall also be recorded in the exception log,
including documentation of the issues, the results of any investigations of these issues, any
corrective actions taken, and pertinent communications, with sign-off by the laboratory director
or designee.
④ The exception log is also required to retain links to the patient reports, and the laboratory
director can choose to communicate any clinically relevant SOP deviations to the ordering
physician. (See 4.4.1 e).)⑤ Exception log documentation can also be incorporated into the monthly quality assurance
report.
⑥ Deviations, such as needing to rerun the analytic pipeline due to network, computer, storage
failure, or memory issues, in which a particular step is performed with different parameters or
cutoffs than those used to validate the assay shall be documented along with the final report andexplanation.
For example, a laboratory can need to alter settings on specific tools or components of its
bioinformatics pipeline to adequately analyze particular regions or variants in a given patientcase.
⑦ The reason for the deviation shall be described in the exception log, as well as the specificcomponents of the deviation. Each deviation should be linked to the associated patient case andreviewed by the laboratory director or appropriate design (s). The deviation or aspects of it canbe included in the final report or in specific communication with the ordering physician
according to agreements.
⑧ Deviations related to bugs or failures in the bioinformatics pipelines also need to be recorded in
the exception log.
⑧ The bug, affected cases, and proposed corrective action shall be approved, signed, and dated by
the laboratory director or designee.
⑨ Outright failures of the bioinformatics pipelines, which might have resulted from hardware aswell as software or operator error, shall also be recorded in the exception log to document errorsthat can have occurred in analyzing individual patient cases.
⑩ Evidence of compliance for the exception log requires the ability to demonstrate appropriate
documentation of review of the exception log by the laboratory director, demonstration that thelaboratory records any issue arising during the bioinformatics procedure, and adequate
documentation of subsequent corrective actions taken as a result of these reviews.
4.10 Corrective Action
・ The program addresses common problems that arise during testing. “Problems” include events
that can affect the test result or its clinical use, as well as nonconformities with the laboratory’s own
policies and procedures. Documentation includes both a review of the effectiveness of corrective
ISO 15189 Application Guidance Document for Molecular-Genetic Examination13

actions taken and the revision of policies and procedures intended to prevent recurrence.
4.11 Preventive action＊
4.12 Continual improvement＊
4.13 Control of Records
Laboratory Records
Methods, instrument(s), and reagents used for processing and analyzing samples (specimens) (orbatches of samples) can be identified and traced in the laboratory records.
① Comprehensive records of laboratory assay ‘‘runs’’ are essential to document the conditions andevents associated with the complex processes and algorithms involved in the performance andinterpretation of NGS-based analyses. Accordingly, such archived information shall bemaintained within a comprehensive framework where all reagents, primers, sequencing
chemistries, and platforms used for the analysis of each patient sample are traceable.
② Such records shall contain a description of the test performed, including the nature of the target
sequence (e.g. genome, exome, specific genes for target panels, transcriptome, or methylome)and depth of coverage (e.g. range and average).
③ It is also necessary to cite details of the analysis, including any publications or websites (withdates accessed) describing the pertinent parameters or other information and/or notations
relative to the testing and reporting processes.
④ While all details of the analysis need not be included in the patient report, it is critical that thelaboratory maintain a documentation system from which detailed information regarding theanalysis of individual patient specimens can be obtained.
Data Storage
The laboratory shall have a policy regarding the storage of input, intermediate, and final data files
generated by the bioinformatics pipeline.
① The laboratory shall establish and follow a procedure for the storage of data files generated by
the bioinformatics pipeline.
② Large data files are generated by NGS and the associated data analysis, including flow cell
imaging files, sequence read files containing base calls and associated quality scores, otherintermediate files generated after subsequent analysis steps, and variant text files.
③ It is generally not practical to retain all such files for an extended period, so this checklist
requirement mandates that the laboratory establish a policy for data storage that specifies data
file retention times and which files will be retained after a final report has been generated. It is
recommended that, if feasible, the laboratory retain sequence files with corresponding quality
scores (e.g. FASTQ file) or retain an archival format from which these files can be regenerated
14

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

(e.g. BAM file).
④ For genome or other large-scale sequencing data, retention of FASTQ files or standard archival
formats for long periods of time can be cost-prohibitive with current storage technologies. The
period for which such files shall be stored is a more complicated decision that depends on
numerous issues, including the size of the data set, laboratory storage capacity, and medicolegal
considerations, as well as other institutional, local, or national requirements for data storage.
⑤ It is emphasized that the laboratory’s policy for data storage and file retention times should be inaccordance with local and national requirements for data storage.
・ The retention period of all information related to reports should be at least five years in accordancewith the Medical Care Act, but as long as possible, depending on the circumstances of thelaboratory and technical requirements.
h)examination results and reports.

・ The laboratory records and maintains all information related to the report. The period should be
at least five years in accordance with the Medical Care Act, but as long as possible, depending on
the circumstances of the laboratory and technical requirements.
4.14 Evaluation and Audit
4.14.4 Staff Suggestions
・ The program should also encourage laboratory personnel to communicate concerns about thequality of laboratory testing. The investigation of personnel complaints and suggestions is part of
the quality assurance program.
4.14.7 Quality Indicators
・ Quality indicators can be specified and defined based on the validation process and results.
4.15 Management review＊

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

15

5

Technical Requirements

5.1 Personnel
5.1.1 General・ The laboratory that performs molecular examination and a place that performs similar tests shallestablish a system that guarantees genetic expertise. This is equivalent to that applied in other areasof clinical laboratory testing. In addition, it includes a system to confirm the validity of requirements
for education/training, skills/qualifications specific to molecular examination, and procedures toconfirm the validity of requirements for testing equipment and operations.
5.1.2 Personnel Qualifications
・ The laboratory shall be directed by personnel with clear qualifications and responsibilities regarding
the services provided.
Laboratory Director
Licensed Doctor of Osteopathic Medicine or Orthopedic Surgery (DO; Medical qualification
similar to MD in the United States) or Doctor of Preventive Medicine (DPM) and certification orequivalent in anatomical or clinical pathology. Or trained residency/fellowship in a medicallaboratory for at least one year, or at least two years of experience supervising high-complexity
testing.
【Note 1】・ FDA defines “high-complexity testing” as the scores (e.g. knowledge, training and experience,
preparation of reagents and materials, and characteristics/automation/QC in operational steps).
・ Evidence of Compliance;
Resume, degree, license, certificate by expert advisors, records of education and training, and work
experience in related fields.
【Note 2】・ The “laboratory director” corresponds to ISO 15189.
・ The “manager” of the registered clinical laboratory and the “responsible persons for ensuring
accuracy” of medical institutions shall be medical doctors or clinical laboratory technicians.
・ In the US, medical genetic doctors signs the final report.
・ Assignment of a person responsible for ensuring accuracy: When conducting molecular-genetic
examination, a person responsible for ensuring accuracy shall be appointed. In addition, it isappropriate to allow a responsible person to ensure the accuracy of the overall testing and a
responsible person to ensure the accuracy of molecular-genetic examination to serve concurrently.
Responsible for ensuring accuracy of molecular and cytogenetic testing①

16

A manager or a responsible person to ensure accuracy can concurrently serve.

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

【Note 1】
・ The practical experience required for the responsible person to ensure accuracy of molecular andcytogenetic testing can be met in at least three years by performing examination and quality
control operations at the same time.
【Note 2】・ It is desirable that the person responsible for ensuring the accuracy of molecular and cytogenetic
testing be a full-time employee, but the laboratory can decide whether or not the responsible
person is to be a full-time employee in consideration of its actual situation.
② Examples of a person who has expertise and experience in molecular and cytogenetic testing
shall include those who meet the following conditions: with more than three years’ experiencein examination and more than three years’ experience in quality control (if served concurrently,
their experience can be applied to meet both requirements despite the overlap), and who havetaken molecular biology-related subjects (such as molecular biology, genetic analysis, cytogenetics,
human genetics, microbiology, biochemistry, immunology, hematology, physiology, anatomic
pathology, anatomy, animal cell engineering, or biological science) at graduate schools,
universities, colleges, vocational schools, or colleges of technology.
③ When a medical doctor or a clinical laboratory technician is appointed as the person responsible
for ensuring the accuracy of molecular and cytogenetic testing, it is desirable to refer to ②
above.
・ The management system for conducting molecular and cytogenetic testing and the quality
assurance system should be established and maintained. ① In order to provide appropriate testing
reports and related information in response to requests from customers, not only to conduct theessential confirmation but also to instruct and supervise testing persons in charge. ② To strive toensure appropriate accuracy by sharing with the person responsible for quality assurance. ③ To
conduct proper assignment based on the competency performance of testing persons in charge
and provide continuous education, training, and skill evaluation.
・ Appropriate experience and capabilities required for the responsible person: For the capability, it
is appropriate to have expertise in clinical testing, including molecular examination. As a generalrule, it is desirable to have a certain amount of experience as practical experience related tomolecular examination, following the requirements of the quality control manager at the registeredclinical laboratory.
・ The person responsible for ensuring accuracy in molecular examination (medical doctor/clinicallaboratory technician) should have appropriate experience and capability. For experience, as a
general rule, it is desirable to require a certain level of practical experience related to molecularexamination. For capability, it is necessary to have expertise in clinical testing, including molecularexamination.
・ In the case of dental institutions, the person responsible for ensuring the accuracy of molecular
ISO 15189 Application Guidance Document for Molecular-Genetic Examination17

examination shall be a dentist or a clinical laboratory technician. In the case of medical institutions,the head of the department that conducts clinical laboratory tests does not necessarily have to be
responsible for ensuring the accuracy of the overall testing.
・ The person responsible for ensuring the accuracy of clinical laboratory tests other than moleculargenetic tests can concurrently serve.
・ The occupation of the responsible person to ensure accuracy of registered clinical laboratory tests
shall be a medical doctor or a clinical laboratory technician (a dentist or a clinical laboratory
technician at a dental medical institution, and a midwife at a maternity hospital). Although nospecial requirements have been set for practical experience, it is advisable to refer to the requirements
for the quality control manager in the registered clinical laboratory (appoint a person with morethan 6 years of practical experience related to clinical laboratory tests and more than 3 years ofpractical experience related to quality control).
【Note 3】・ If there is no suitable medical doctor or clinical laboratory technician at a medical institution, thisshall not prevent people in other occupations who have expertise and experience in moleculargenetic examination from becoming the head of the department that conducts clinical laboratory
tests.
・ A person responsible for ensuring accuracy shall be appointed. In addition, under the law thatpartially amended the Medical Care Act, the responsible person to ensure the accuracy of allclinical laboratory tests is allowed to serve concurrently. Regarding capability, expertise in quality
control of all clinical testing (including clinical laboratory tests), including molecular examination
and appropriate experience and capability, is required.
【Note 4】・ Familiarity with the quality control of both clinical testing and molecular examination is desirable.
Technical Supervisor
A pathologist and/or a medical doctor (MD) or a doctor of osteopathy (DO) certified as a clinicalpathologist, or possess qualifications equivalent to those required by a panel of experts (e.g. medical
doctors). The technical supervisor, delegated by the laboratory director, is responsible forsupervising all laboratory practices, including technical and scientific aspects, and for conducting
and recording competence assessments in high-complexity testing.
Evidence of Compliance・ Records of qualifications, including diploma, transcript(s), or current license, if required.
・ Certification/registration.
【Note】・ This corresponds to the “technical personnel” of ISO 15189.
・ The qualification of an anatomic pathologist is a clinical pathologist in Japan, and the qualificationof a clinical pathologist is a licensed doctor of a clinical laboratory physician.
18

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

・ High-complexity testing refers to anything other than a molecular examination system consistingof a series of pharmaceutically approved reagents and equipment. Examples include pathological
specimens, multiplex analysis, and sequencing.
Clinical Consultant
・ Medical doctor or holder of a PhD in science. The details of this are described below.
Evidence of compliance・ Qualification record of clinical consultant.
General Supervisor・ Medical doctor, holder of a PhD in science or of a bachelor’s degree in a chemical, physical,
biological, clinical laboratory science, or medical technology with at least one year of experiencein high-complexity testing; orAssociate degree in laboratory science or medical technology with at least two years of experiencein high-complexity testing; orLaboratory director or technical supervisor of a laboratory that performs high complexity testing,or medical doctor (MD), doctor of osteopathy (DO), doctor of preventive medicine (DPM),
PhD, master’s degree, or bachelor’s degree holder in clinical laboratory science with at least one
year of experience in high-complexity testing.
Evidence of compliance・ Records of qualifications, including diploma, transcript(s), or current license.
・ Certification/registration.
【Note 1】・ An associate degree is a clinical laboratory technician who has graduated from a vocational school.
・ The manager of the molecular department shall be properly educated and trained to ensure thequality of the department’s molecular examination.
At the least, the manager of the molecular department should have the following expertise andskills:① can consider whether the request for testing is appropriate,② can confirm the validation of the testing,③ can perform the examination and interpret the results,④ can communicate to the medical institutions about the testing result and related information,
whether expert or non-expert,⑤ can take responsibility for the operation of molecular examination facilities on a daily basis, and⑥ can establish and maintain a quality control system.
Personnel in the molecular department
For testing that requires advanced medical expertise and skills, in order to ensure the appropriatenessof the testing, it is desirable that those who have expertise and skills such as clinical laboratory
technicians perform the testing.
ISO 15189 Application Guidance Document for Molecular-Genetic Examination

19

【Note】
・ The main qualifications related to molecular examination technologies are as follows:
Certified qualifications for molecular and cytogenetic testing include Genetic Analysis Science
Certification (beginner/first grade) (Japan Institute of Genetic Analysis Science), ClinicalCytogenetics Certification (Japan Society of Human Genetics), certified qualifications for ClinicalCytogenetics (Japanese Society of Cytogenetics/Japanese Association of Medical Technologists),
Biotechnologists (Japan Association of Biotechnology Education), Genetic Experts (JapaneseSociety for Gene Diagnosis and Therapy), and Bioinformatics Engineers (Japanese Society for
Bioinformatics).
・ Examination should be performed by bachelor-level personnel, personnel recognized as equivalent
in the laboratory, or personnel under their control. In addition, if relevant professional qualifications
are required, they shall be done by qualified and experienced personnel.
・ Personnel certified as bachelor-level are usually considered to be experienced personnel with about
two years of relevant operation experience.
・ Personnel who are undergoing education/training or do not have the relevant qualifications canconduct an examination if they have been clearly educated/trained at the appropriate level andhave appropriate supervision.
5.1.5 Training
・ The laboratory should recognize that education and training in clinical genetics is essential for
conducting molecular examination.
・ Testing department managers should ensure that personnel conducting molecular examination
continue to participate in education and training programs.
b)assigned work processes and procedures・ The laboratory shall ensure that all personnel are educated and trained to carry out examinations
and operate equipment properly.
・ Appropriate education/training standards and professional qualifications should be established for
persons in charge of the molecular department. Educational requirements should include
systematic training in molecular biology, molecular genetics in clinical tests, and other specialized
subjects in related fields.
c)the applicable laboratory information system

・ All related personnel to operate the laboratory information system shall be trained initially, after
system modification, and after the installation of a new system.
d)health and safety, including the prevention or containment of the effects of adverse incidents

・ Training (education/training) on understanding the concept of biosafety based on biorisk
evaluation shall be implemented.
e)20

ethics;ISO 15189 Application Guidance Document for Molecular-Genetic Examination

・ Training (education/training) to understand ethics shall be implemented.
【Note】・ Ethics shall include medical ethics: International declarations and agreements on medical ethics
including the “World Declaration on Human Genome and Human Rights,” “International
Declaration on Human Genetic Information,” “World Declaration on Bioethics and Human
Rights,” “Guidelines for Genetic Tests and Diagnosis in Medical Practice” (The Japanese
Association of Medical Sciences. February 2011), “Ethical Guidelines for Human Genome/
Genetic Analysis Research” (Ministry of Education, Ministry of Health, Labor and Welfare,
Ministry of Economy, Trade and Industry), “Ethical, Legal and Social Implications” (CDC:
American Center for Disease Control and Prevention), and “Ethical Guideline for Medical
Research in Humans” (Ministry of Education, Culture, Sports, Science and Technology, Ministryof Health, Labor and Welfare, Ministry of Economy, Trade and Industry).
f)Confidentiality of patient information

・ The patient’s personal genetic information shall be protected in accordance with the Personal
Information Protection Law and guidelines based on it.
・ To properly protect the personal data handled, it is necessary to raise awareness about the protectionof personal information through education and training for personnel.
・ It is necessary to consider the implementation of education and training regarding the handling of
personal information, based on the guideline that “for temporary workers, the laboratory should
also strive to provide education and training as needed.”
5.1.6 Competence Assessment
・ The laboratory should evaluate the degree of skill acquisition during education and training using
an objective scale. For example, the laboratory shall monitor that personnel competencies havebeen maintained using quality control techniques.
【Note】
・ The laboratory director shall ensure that all testing personnel are properly trained and haveappropriate competencies before performing molecular examinations.
f)examination of specially provided samples, such as previously examined samples, interlaboratorycomparison materials, or split samples.

【NOTE】・ NMIJ CRM 6204-b and 6205-a are examples of interlaboratory comparison substances (seeSection 5.6.3.2).
5.1.8 Continuing Education and Professional Development
・ The laboratory manager is responsible for ensuring proper education and training. The importanceof self-edification should be emphasized, especially for expert analysts.
ISO 15189 Application Guidance Document for Molecular-Genetic Examination

21

5.1.9 Personnel Records
・ The laboratory shall maintain the latest records of education and training conducted by personnel.
Records typically include the following:a)educational and professional qualifications

・ Educational background and professional qualifications;b)copy of certification or license, when applicable:

・ Transcript, certification/registration certificate;f)training in current job tasks:

・ Records of attendance of training courses inside and outside the laboratory,・ Related OJT and re-education/re-training conducted as needed;h)records of continuing education and achievements:

・ Published technical papers and presentations in academic societies.
5.2 Accommodation and Environmental Conditions
5.2.1 General・ It is necessary to establish a safety management plan for pathogens based on biosafety and strive
to prevent unintentional exposure or leakage of pathogens and toxins (see Section 5.2.6).
5.2.2 Laboratory and Office Facilitiesa)

Access to areas affecting the quality of examinations is controlled.

・ Depending on the operation to be carried out, it can be necessary to restrict access to specific areasof the laboratory. If restricted access is in effect, personnel should be informed of the following:① Purpose of using a specific area
② Operational restrictions on the area.
③ Reasons for the restrictions④ Actions taken when such restrictions are violated.
・ The laboratory shall establish a safety management plan for pathogens based on the concept ofbiosafety and strive to prevent unintentional exposure or leakage of pathogens and toxins.
b)

Medical information, patient samples, and laboratory resources are safeguarded from unauthorizedaccess.

・ To prevent theft or loss of personal data, the laboratory shall take the following physical security
management actions:・ Entrance/exit (room) management, preventive actions against theft (for example, filming with acamera/video), and physical protection such as fixing of equipment and/or devices.
・ Based on the operational needs to prevent unauthorized operations, the functions given toterminals that handle personal data are restricted as follows: connection restrictions and response
to device updates for devices with recording functions such as smartphones and personal computers.
22

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

e)

Safety facilities and devices are provided and their functioning regularly verified.

・ During UV irradiation, UV-blocking protective glasses or protective shield glass shall be used toprevent direct exposure of the eyes to UV rays.
5.2.6 Facility Maintenance and Environmental Conditions
・ Deviations from the limits of environmental conditions can be identified by monitoring thesystem or quality assurance of certain analyses. The impacts of deviations from the environment
can be assessed as part of the robustness test through validation. Emergency operating proceduresshould be established as appropriate.
・ All temperature-controlled equipment and environmental conditions shall be checked daily.
・ In the biobank, there shall be evidence that samples (specimens) are stored at the temperature
required by the protocol.
・ In the biobank, the temperature shall be checked daily with thermometers for all temperaturedependent equipment and environment.
・ In the biobank, when the temperature is out of the acceptable range, appropriate corrective action
should be taken, and then the temperature should be checked again and recorded.
・ The identity of the individual recording the temperature(s) shall be documented (recording theinitials of the individual is adequate).
・ The use of automated (including remote) temperature monitoring systems is acceptable, and thefunctionality of the system shall be documented daily.
・ In the biobank, temperature-controlled storage equipment shall have an emergency power supply,
if necessary.
・ There are documented procedures to follow if there are deviations in the storage temperature
limits.
・ Alarm systems will continue to function if power is interrupted.
・ Temperature-acceptable limits for the alarm system are established considering the anticipated
response time.
・ Molecular examination laboratories should be divided into at least two areas, a nucleic acidextraction/amplification reagent preparation area and an amplification/detection area, to avoid
contamination by nucleic acid amplification products. In the case of fully automated molecularexamination equipment, it is necessary to ensure that contamination measures are taken according
to the internal structure of the equipment.
・ A clean bench A clean bench that can keep the inside clean should be used in the nucleic acidextraction/amplification reagent preparation area. A clean bench shall be used as the area of reagent
preparation for amplification reagents.
・ Under the Act on Clinical Laboratory Technicians (before the 2018 amendment), registeredclinical laboratories that carry out molecular examination in the classification of clinical laboratory
ISO 15189 Application Guidance Document for Molecular-Genetic Examination23

tests are required to have a safety cabinet as machinery and equipment for testing.
・ If the room cannot be divided, the laboratory should separate the nucleic acid extraction area andthe amplification reagent preparation area using a desktop biosafety cabinet with ultraviolet rays.
If there is no hood equipped with an ultraviolet irradiation device, it is also effective to change themeasurement location and wipe the laboratory bench top and pipette thoroughly with a 0.5%
sodium hypochlorite aqueous solution before and after use.
・ Pipettes, tips with filters, and tubes shall be dedicated to each area and irradiated with ultraviolet
rays to destroy DNA. It is desirable to prepare two sets of pipettes, one for the reagents and one
for the samples.
【Note】
・ Use a clean bench when preparing reagents and a biosafety cabinet when handling infectious
specimens. The biosafety cabinet requirement was removed from the amended Act on ClinicalLaboratory Technicians.
・ When selecting a designated area for a new operation, previous use of that area should beconsidered.
5.3 Laboratory equipment, reagents, and consumables
5.3.1.2 Equipment Acceptance Testing・ The laboratory shall perform the following applicable activities to verify that the equipment meetsthe required specifications and any other specifications related to the testing.
Installation Qualification (IQ)
After the equipment is installed in the actual operating location, it is necessary to verify anddocument whether the equipment configuration is appropriate and whether the equipment,
including safety features, is installed correctly. The installation qualification and operator training
for all equipment used for testing should be completed.
Operational Qualification (OQ) and Performance Qualification (PQ)
It is necessary to verify that the equipment and its measurement subsystem meet the required
functional and performance specifications through various tests.
【Note】 OQ and PQ shall be carried out on a regular basis.
5.3.1.4 Equipment Calibration and Metrological Traceabilityb)recording the metrological traceability of the calibration standard and the traceable calibration of
the item of equipment.

【Note】
・ The laboratory shall have a thermometer with a calibration certificate (calibration certificate with
JCSS mark issued by JCSS calibration company, calibration certificate with international mutual
approval MRA marked, calibration with calibration certificate by NIST or PTB) used to measure24

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

temperature as the standard.
d)recording the calibration status and date of recalibration.

・ Equipment that requires calibration, such as temperature measuring devices, shall be recalibrated
and recertified prior to the date of expiration of the guarantee of calibration; documentation ofrecalibration/recertification shall be maintained for review.
e)ensuring that, where calibration gives rise to a set of correction factors, the previous calibration
factors are correctly updated.

・ An appropriate calibrated thermometer shall be used for measuring temperature.
【Note】・ All non-certified thermometers shall be checked against an appropriate thermometric standard
device guaranteed by the JCSS calibration certificate, MRA marked calibration certificate, andNIST or PTB calibration certificate before initial use.
・ The laboratory should work with relevant national and international institutes to collect, develop,
validate, and make available reference standards for molecular-genetic examination.
・ The laboratory should use reference standards and family specific mutation references wheneveravailable.
g)safeguards to prevent adjustments or tampering that might invalidate examination results.

・ The laboratory should work with relevant national and international institutes to collect, develop,
validate and make available reference standards for molecular-genetic examination.
・ The laboratory should use reference standards and family-specific mutation references whereveravailable.
【Note】
・ Although it is a “standard material” in the literature, standard materials have not been developed
for many tests. Therefore, it is paraphrased as “reference standard.”
5.3.1.5 Equipment Maintenance and Repair【Note】・ For equipment maintenance, refer to JAB RL358 Annex A2, guidelines for equipment validation,and performance verification.
・ For unmodified manufacturers’ devices, equipment, or testing systems, the laboratory shallconduct the following:① The laboratory shall perform and document the maintenance specified by the manufacturer.
② The laboratory shall perform and document the functional checks specified by the manufacturer
at the specified frequency. The functional confirmation shall be within the tolerances set by the
manufacturer before performing the patients’ tests.
・ For the instruments, equipment, and testing systems developed by the laboratory, commerciallyavailable, and modified by the laboratory, the laboratory shall conduct the following if maintenanceISO 15189 Application Guidance Document for Molecular-Genetic Examination25

and functional verification protocols are not provided by the manufacturer:① The maintenance protocols for instruments, equipment, or testing systems required for accurate
and reliable testing results and testing result reporting shall be established, implemented, anddocumented.
② The functional verification protocols that ensure the performance of instruments, equipment, or
testing systems required for accurate and reliable testing results and testing result reporting shallbe defined, implemented, and documented. Functional verification shall be within theestablished tolerances of the laboratory before performing the patients’ tests.
5.3.2.7 Reagents and Consumables — Records
・ Prepared reagents and reference standards shall be labeled to indicate substance name, concentration,
solvent (if not purified water), special precautions or hazards, expiration date, preparation date,
and/or expiration date. The person responsible for preparing the reagent shall be identified fromthe label or record.
【Note】
・ Although it is a “standard material” in the literature, standard materials have not been developed
for many molecular-genetic tests. Therefore, it is paraphrased as “reference standard.”
・ A reference standard is defined as “a substance that is sufficiently homogeneous and stable withrespect to one or more specific characteristics established to suit the intended use in themeasurement process.”
・ The characteristics shall be determined using different procedures to minimize the bias relevant toa particular method. For NGS, different NGS techniques or characterization using orthogonal
methods, such as Sanger sequencing and quantitative real-time PCR (qPCR), can be required. In
this way, consensus values, such as genotypes, can be assigned to the reference standard.
・ To fit the intended use, it shall function as well as the samples (specimens).
・ Commutability requires clinical DNA standards to react similarly to a patient’s genomic DNA(gDNA) during library preparation, sequencing, and analysis. Using reference standards with lesscommutability causes biases in the calibrated measurement values and makes the diagnosis
inaccurate. Once commutability is established, reference standards can be used to calibratemeasurements from patient specimens. The uncertainty of DNA sequences in patient specimens
can be determined by comparison with the reference standards for known abundances, along withthe uncertainties relevant to this measurement. This makes it possible to standardize themeasurement values across multiple specimens and fix the diagnostic threshold to the reference
standards.
5.4 Pre-examination processes
5.4.2 Information for Patients and Users
26

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

g)

instructions for patient-collected samples.

・ The person responsible for sample collection shall be informed of how to handle and store thevarious collected samples (specimens).
l)availability of clinical advice on ordering of examinations and on interpretation of examination
results.

・ Molecular examination laboratory should disclose information about the characteristics andlimitations of the tests provided, including analytical validity, clinical validity, and usefulness.
・ The laboratory facilities should ensure that service users understand the latest evidence of theclinical validity and usefulness of the provided tests.
n)the laboratory’s complaint procedure.

・ The laboratory shall ensure that consent is obtained from the patient with sufficient explanation
regarding the implementation of the test and before and after the test.
・ If necessary, medical doctors, licensed doctors of clinical geneticists, or licensed doctors of clinicallaboratory specialists, and personnel (such as pharmacists, nurses, and clinical laboratory
technicians) with expertise in pharmacogenomics testing shall explain the procedure to thesubjects.
・ There shall be an explanation procedure for the subject when conducting molecular-genetic tests.
・ Because the items to be explained to the subject differ depending on the product of thepharmacogenomics testing and the content of the testing, information on the characteristics of the
product/testing shall be obtained from the providing company (diagnostic drug manufacturer andregistered clinical laboratory). The same information shall be provided to the subject when amedical institution performs tests using the home-brew method in its own facilities.
・ The sample provider shall notify the biobank on how to share the obtained data.
・ There shall be procedures and records of informed consent when conducting molecular-genetic
tests.
・ Documents include the following: ① period of storage of specimens; ② management of residual
specimens; ③ disposal method; ④ possibility of re-examination with specimens (for example, due
to significant advances in knowledge and technology), ⑤ possibility of secondary use underanonymity for the purpose of quality control, ⑥ possibility of access to the specimen by a third
party, and ⑦ confidentiality protection methods (symbolization/anonymization).
【Note】
・ International declarations and agreements on informed consent include the “Universal Declarationon Human Genome and Human Rights,” “International Declaration on Human Genetic
Information,” and “Universal Declaration on Bioethics and Human Rights.”
・ The analysis results found in the next-generation sequencer include the “primary findings” that are
the main purpose of the test and “secondary findings,” as described below. The main purpose of
the test needs to be explained in detail to the subject with sufficient time provided for clarification,
ISO 15189 Application Guidance Document for Molecular-Genetic Examination27

but it is also necessary to provide an explanation beforehand and get understanding about
secondary findings.
5.4.3 Request Form Informatione)clinically relevant information about the patient and the request, for examination performanceand result interpretation purposes.

・ The laboratory shall confirm that the testing request is appropriate, and if necessary, inform thesubject that information on the subject/family is required to correctly interpret the test result.
【Note】
・ The significance of test results often depends on the accuracy and appropriateness of the information
provided to the laboratory. All information necessary to carry out the testing, including the statusof transportation of the samples (specimens) to the laboratory, shall be sent to the laboratory withthe samples (specimens).
5.4.4 Primary Sample Collection and Handling
5.4.4.1 General・ According to the purpose of the testing, the procedures of collection and storage to ensure quality
for each type of primary sample (specimen) should be clarified.
【Note 1】
Typical primary samples are shown according to the purpose of testing.
① Nucleic acid tests for pathogens: serum, plasma, urine, sputum, feces, etc.
② Molecular-genetic tests for human somatic gene alterations: tissue, blood (white blood cells),
plasma, bone marrow, urine (sediment), sputum③ Human genetic testing: blood (white blood cells), oral mucosa, hair, nails, blood stains,umbilical cord
【Note 2】
Recommendation (draft): Report of a test result to patients using stored samples in a biobank.
① Report of a test result to the patients using biobank samples is premised on a quality assurance
mechanism.
② There shall be documented policies and procedures to ensure the quality of the sample.
③ Refer to the following for the quality control and assurance of biobanks for clinical use:
Domestic standard documents (such as Japanese Society of Pathology, JCCLS);
International standards: ISO 15189 standard documents related to pre-measurement
processes, ISO 20658 related to collection, ISO 20166, ISO20184, ISO 20186, and ISO
21474 related to specimen/nucleic acid (ISO 15189 guidance document).
④ An anatomical laboratory with a clinical biobank should undergo a bank function audit by
third-party accreditation (e.g. ISO 15189) as a mechanism for quality assurance.
28

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

⑤ When returning samples and information to patients at the research biobank, it shall beconsidered whether or not to comply with 2 and 3 above, how to deal with them, and reflect
on the report (e.g. impact on the test results, test limits) in cooperation with the third-party
accredited laboratories specified in 4 above.
・ In genetic testing for the purpose of diagnosing monogenic diseases, personal information shall beprotected by anonymization and confidential reporting.
5.4.4.2c)

Instructions for Pre-Collection Activities

type and amount of the primary sample to be collected with descriptions of the primary sample
containers and any necessary additives.

・ The containers of the primary samples (specimens) for molecular-genetic tests and necessary
additives shall be appropriately selected according to the purpose of the tests (nucleic acid tests forpathogens, molecular-genetic tests for human somatic gene alterations, human genetic test), andsample type (e.g. blood, plasma, urine, and tissue).
d)special timing of collection, where needed.

・ When collecting liquid-based cytology specimens, an appropriate collection method that cansufficiently collect the target cells shall be specified and explained.
e)clinical information relevant to or affecting sample collection, examination performance or resultinterpretation (e.g. history of administration of drugs).

・ While the intended use of specimens is not always known, the specimens shall typically be stored
for use in a wide range of molecular-genetic tests based on an understanding of the variable factorsthat affect the quality of the specimen.
・ In the cytogenetic testing of tumor cells, there shall be instructions on the timing of sample(specimen) collection, such as collection before administration of anticancer drugs.
5.4.4.3 Instructions for Collection Activitiesb)

verification that the patient meets pre-examination requirements [e.g. fasting status, medication
status (time of last dose, cessation), sample collection at predetermined time or time intervals,etc.].

・ In nucleic acid tests for pathogens (virus tests), it is important to collect appropriate specimenmaterials at the appropriate stage in order to perform the testing accurately.
c)

instructions for collection of primary blood and non-blood samples, with descriptions of the
primary sample containers and any necessary additives.

・ The containers of the primary samples (specimens) for molecular-genetic tests and necessary
additives shall be appropriately selected according to the purpose of the tests (nucleic acid tests forpathogens, molecular-genetic tests for human somatic gene alterations, human genetic test), andspecimen type (e.g. blood, plasma, urine, and tissue).
ISO 15189 Application Guidance Document for Molecular-Genetic Examination

29

d)

in situations where the primary sample is collected as part of clinical practice, information andinstructions regarding primary sample containers, any necessary additives and any necessary
processing and sample transport conditions shall be determined and communicated to theappropriate clinical staff.

・ The container of the primary samples (specimens), necessary additives, and transportation
conditions shall be documented, specified and explained.
e)

instructions for labelling of primary samples in a manner that provides an unequivocal link withthe patients from whom they are collected.

・ Specimen storage containers should be uniquely identified and managed using barcodes (1D, 2D,etc.) or RFID to accurately track and guarantee specimens. When barcodes are unique and easy toread, they are expected to automate many downstream processes and avoid the risk of humanerror.
g)

instructions for proper storage conditions before collected samples are delivered to the laboratory.

・ The person responsible for specimen collection shall be notified of how to handle and store thevarious collected specimens.
・ The appropriate storage conditions (temperature and storage container) for the primary samples(specimens) for molecular-genetic tests shall be established. The extracted RNA is particularly
unstable. The storage containers shall be considered to have the potential for the adsorption of
nucleic acids.
5.4.5 Sample Transportation
・ The appropriate transport methods and transport temperature control (freezing, refrigeration,
room temperature) shall be established and documented according to molecular-genetic test
samples (specimens).
b)

within a temperature interval specified for sample collection and handling and with the designatedpreservatives to ensure the integrity of samples.

・ The appropriate storage conditions and storage temperatures for samples for molecular-genetic
tests shall be established.
5.4.6 Sample Reception
・ The received sample shall be evaluated to determine whether it is suitable for the intended testing.
a)Samples are unequivocally traceable, by request and labelling, to an identified patient or site.

・ The history of specimen collection, storage, and transportation shall be traceable.
b)

Laboratory-developed and documented criteria for acceptance or rejection of samples are applied.

・ The criteria for rejection of samples (specimens), such as confirming inappropriate collection
containers or storage conditions that affect the results, shall be established.
d)All samples received are recorded in an accession book, worksheet, computer or other comparable

30

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

system. The date and time of receipt and/or registration of samples shall be recorded. Wheneverpossible, the identity of the person receiving the sample shall also be recorded.
・ The receipt of samples shall be recorded reliably.
5.4.7 Pre-Examination Handling, Preparation, and Storage
・ There shall be a documented procedure to follow if there are deviations in the storage temperature
limits.
・ When a medical institution refers molecular-genetic tests using a pathological specimen to aregistered clinical laboratory, the medical institution can gross the specimen or the registeredclinical laboratory can gross it. In the case of grossing in a registered clinical laboratory, when the
medical institution submits the pathological specimen to the registered clinical laboratory, themedical doctor shall give specific instructions on the part to be grossed. Based on this, the registeredclinical laboratory shall make the grossing and confirm that the grossing is performed according
to the instructions.
5.5 Examination processes
5.5.1.1 General・ The laboratory shall select a measurement procedure that meets users’ needs, including sampling
methods, and is suitable for the testing performed. The laboratory shall select one of the appropriate
methods published as international standards, regional or national standards, publications of wellestablished technical institutions, and relevant scientific literature or periodicals.
5.5.1.2 Verification of Examination Procedures【Note】・ NGS-based tests regulatory-approved by pharmaceutical affairs correspond with this section.
・ The following verification is required before reporting the test results to the patient.
Accuracy
・ Closeness of agreement between the measured quantity value including the accuracy and precisionand the true quantity value of the measurand.
・ The definition in NGS is the closeness of agreement between the nucleic acid sequence derived
from the assay and a reference sequence.
Precision・ Closeness of agreement between indications or measured quantity values obtained by replicate
measurements on the same or similar objects under specified conditions. Measurement precision
is usually expressed as a measure of imprecision; that is, it is calculated as the standard deviationof the measurement.
・ The definition in NGS is the closeness to which repeated sequence analyses give the sameISO 15189 Application Guidance Document for Molecular-Genetic Examination31

repeatability and reproducibility.
Reportable range
・ The definition in NGS is the region of the genome in which sequences of acceptable quality canbe detected by testing.
Reference range
・ The definition in NGS is the range of base sequence variations detected in the unaffected
population.
5.5.1.3 Validation of Examination Procedures
・ Platform validation
・ Test validation: Disease-associated variants are correctly identified in targeted regions of the
genome.
【Note】・ Validation is conducted to confirm by investigation that individual requirements are adequate fora specific intended use and to provide objective evidence.
・ The basis for validation is the establishment of a reliable sequence analysis across the genomic
regions targeted by the test.
・ Test validation is conducted to establish an accurate identification system that links diseases tomutant genomic regions.
・ Bioinformatics pipeline validation is used to establish the algorithm(s) by reliably analyzing
platform data to produce an accurate sequence.
NGS confirmation testing・ The laboratory shall have a policy that documents indications for confirmatory testing of thereported variants.
① Confirmation tests using other methods can be required to achieve the desired confidence in thevariants that are reported.
② Some laboratories can decide to perform re-evaluation at a later stage and perform confirmation
tests on variants for a predetermined trial period.
③ Each laboratory performing NGS shall have a policy in place that clearly documents indications
for confirmation test and/or documents how they determined that confirmation testing was
unnecessary through their assay validation.
④ The laboratory shall document compliance with their confirmation testing policy and show theevidence of ongoing monitoring of their NGS assay(s) to ensure that the benchmarks achieved
during the validation process are maintained during the routine work of NGS-based clinicaltesting and variant reporting. (See 5.5.1.2.)
・ Measurement systems (e.g. analytical instruments and reagents) used in clinical testing shall be
guaranteed to meet the intended use and to provide reliable results that meet the requirements.
32

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

Therefore, prior to the implementation of panel testing, specifications suitable for the purpose of
the test shall be determined, and instruction manuals, package insert documentation of analytical
instruments, in-vitro diagnostic drugs (template DNA preparation reagents), medical equipment(e.g. DNA sequencer and analysis program), and servers obtained that meet the respective
requirements, various SOPs created, and Installation Qualification(IQ) and Operation
Qualification (OQ) for each equipment performed, as well as Performance Qualification (PQ) as
necessary.
b)laboratory designed or developed methods.

・ The method developed or selected in the laboratory shall be suitable for the intended use and canbe used if validated.
・ The laboratory shall validate the analytical wet-bench process. In addition, if the process is
modified, the entire process should be revalidated and/or confirmation made that the processcomponents perform satisfactorily. The degree of revalidation shall be dependent on themodification. The analytical performance of NGS testing should be internally validated before
clinical implementation.
・ Next-generation sequencing analysis is a complex procedure involving many steps within the wetbench workflow. Each step needs to be optimized individually to empirically determine theoptimal assay conditions and analysis settings. Once those are in place, an analytic validation shallbe performed for the whole test in a ‘‘beginning-to-end’’ fashion, including the entire wet bench
process.
・ Important performance characteristics that need to be determined during the validation are
analytical sensitivity, analytical specificity, accuracy, precision, and limit of detection. For any
molecular assay, validation should also be conducted independently for each received specimen
type.
① Analytical sensitivity
Analytical sensitivity can be assessed using a method-based approach that aims to maximize thenumber of sequence variants that are compared to a gold standard method to increase theconfidence of analytic performance. It is important to determine this baseline performance byusing as many different genomic regions as possible, since sequence context can be an important
influence.
② Analytical specificity
・ Validation requires a combination of analyte-specific methods and methods based on procedure
because NGS permits the detection of novel as well as known sequence variants and necessitates acomprehensive approach to determine test performance with adequate confidence. It is important
to include conventional positive controls with patient samples, including relevant variants foranalyte-specific validation.
・ Analytical specificity is often calculated by using ‘‘negative’’ samples (i.e., samples that have noISO 15189 Application Guidance Document for Molecular-Genetic Examination

33

pathogenic variant) to determine the fraction that is correctly identified as negative.
・ A method-based approach can be leveraged to calculate analytical specificity across the assayedregion, for example, by determining the false-positive rate (fraction of variants detected as incorrect
calls). It is also useful to determine the average number of false-positive calls for the regions tested
in a clinical specimen. Note that the analytical specificity accounts for numerous sources of type I
errors, including base-calling errors, errors due to misalignment, and variant-calling errors.
・ Homologous sequences significantly affect the analysis of affected genes because pseudogenes caninterfere with accurate variant calling. If such genes are included in the NGS test, the laboratory
shall devise a method to ensure that the identified variants are not due to pseudogene sequences
and shall document the accuracy of the method. When pooled sequencing of bar-coded samples
is performed, the laboratory shall document that individual sample identity is maintained
throughout the wet bench process.
③ Precision
Precision (inter-run and intra-run variability) shall be determined using at least three samples.
④ Limit of Detection【Note】
・ Regarding the detection of somatic mutations, the limit of detection can be determined by panel
tests with fixed analysis targets, but it is difficult to determine the range for molecular-genetic tests
for human germline alterations when using genome-wide analysis.
・ Determining the limit of detection is important for assays that interrogate samples withheterogeneous genotypes. Mixing sample experiments can be used to assess the limit of detection
for each variant type.
・ The total number of samples that needs to be run to appropriately validate the NGS test needs tobe evaluated by the size of the test (larger assayed regions include more variants available forderiving their technical performance), by the number of specific analytes (variants) that need to beassessed, by the possible requirement to determine the limit of detection across a range of allele
frequencies, and by the number of runs and samples needed to determine precision.
・ For bioinformatics analysis of NGS data, it is necessary to validate the ① robustness, ② accuracy,
and ③ reproducibility at each step. There are 17 best practice consensus recommendations forvalidating NGS bioinformatics from the Molecular Pathology Association, which is theorganizational representative of the College of American Pathologists and the American Medical
Informatics Association.
Recommendation 1: Medical laboratories should validate their bioinformatics pipeline when
conducting NGS-based testing.
Recommendation 2: Qualified healthcare professionals with appropriate training and certification
in interpreting NGS should be involved in monitoring the validation process.
Recommendation 3: Validation shall be performed after the design, development, optimization,
34

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

and proficiency of the bioinformatics pipeline and component have been completed.
Recommendation 4: Validation of the bioinformatics pipeline should be adapted to the actual
environment of the laboratory in which testing is performed.
Recommendation 5: Validation includes the individual components of all bioinformatics
pipelines used in the analysis. Each component shall be reviewed and approved by a qualified
clinical molecular expert and laboratory director.
Recommendation 6: The design and implementation of the bioinformatics pipeline shall ensure
the security of patient information and comply with all applicable laws and regulations.
Recommendation 7: Validation of the bioinformatics pipeline is appropriate and applicable tothe variants detected by intended clinical use, specimens (samples), and NGS testing.
Recommendation 8: The laboratory shall ensure that the design, implementation, and validationof the bioinformatics pipeline comply with applicable laboratory accreditation standards andregulations.
Recommendation 9: The bioinformatics pipeline is a part of the testing procedure. Its components
and processes shall be documented in accordance with the laboratory accreditation standards andregulations.
Recommendation 10: Specimen identification should be maintained at each step of the NGS
bioinformatics pipeline with at least four unique identification indicators, including location
identification created by the pipeline or characteristics of each datafile read content.
Recommendation 11: Special quality control and quality assurance parameters should be evaluatedfor validation and used to determine satisfactory specifications for the bioinformatics pipeline.
Recommendation 12: Methods of modifying or filtering sequence reads at any point in thebioinformatics pipeline should be validated prior to implementation to ensure that they arepresented in the data interpretation. All these documents are stored as test documents according
to the laboratory accreditation standards and regulations.
Recommendation 13: The medical laboratory should have special measures to maintain the
integrity of each datafile created in the bioinformatics pipeline and to warn or prevent the use ofdata files that have been tampered with or unintentionally modified.
Recommendation 14: The validation of the bioinformatics pipeline can be complemented by insilico analysis, but it cannot be used as a substitute for the validation of the entire bioinformatics
pipeline using human samples.
Recommendation 15: Validation of bioinformatics pipelines includes the confirmation ofrepresentative variant sets with high-quality independent data. Appropriate validation metrics foreach variant type should be reported.
Recommendation 16: The medical laboratory should guarantee the accuracy of the nomenclatureand interpretation of software-created HGVS variants. If they need to be reviewed or correctedmanually, a warning shall appear, and the documents shall be maintained for any corrections.
ISO 15189 Application Guidance Document for Molecular-Genetic Examination

35

Recommendation 17: Supplementary validation is required when significant changes are made to
the components of the bioinformatics pipeline.
・ Validation results will be approved by the laboratory director.
[Note]
SOP (see 5.5.3)
・ Document all steps for sample preparation, data analysis, and judgment.
・ Document the selection and confirmation method of the target of a protocol that confirms
detection.
QC (see 5.6.2)
・ The quality of the base score should be at least 20 or equivalent per base.
・ Establish depth and coverage uniformity, that is, minimum read count, across all target areas. A
minimum of 500 reads is recommended.
・ Define the minimum coverage required for target regions (e.g. amplicon, exons) where the state of
mutation in the region cannot be confidently defined. The minimum coverage required can vary
between mutation and non-mutation.
・ Define the minimum percentage of variant reads in the background of normal reads required tocall a “detected” variant with default reliability and sensitivity.
・ Define the maximum strand bias allowed, if applicable.
・ When changing to a new reagent lot, validation/confirmation of analytical sensitivity is required
to ensure that low positives are not missed with the reagents of the new lot. This applies to all
reagents and includes coating depth and uniformity to detect potential dropout of the target area.
・ All QC metrics should be tracked and documented to ensure that there is no performancedegradation.
・ All software updates that affect critical parameters such as base call and alignment shall bereconfirmed by repeating at least 3–5 runs of previous analyses. This verification also verifies thatthe coverage depth and readability of the variants differ significantly between the two software
versions. The reverse process is clearly documented in the SOP.
Control (see 5.6.2.2)
・ No template control (NTC) shall be included in all amplification steps to ensure that there is nocontamination between the samples and reagents. This control can be analyzed in any suitable way
before proceeding with actual sequencing.
・ Negative controls such as DNA from the HapMap cell line (e.g. NA12878 and/or NA19240)
should be included to verify the accuracy and specificity of the analysis for the detection of
mutations during the initial confirmation and periodically thereafter.
・ Positive/sensitivity controls shall be included in each run. It is suggested that these controls be
individually barcoded low-positive DNA samples containing multiple known variants of each
type, detected near analytic sensitivity to verify that a low percentage of variants is consistently
36

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

identified. If not all variants, and if all target areas can be included in a single control sample, thena defined rotation schedule should be used.
Report (see 5.8.1)
・ The variants detected shall be reported, whether or not the purpose is to detect all somatic variants,
whether the clinical significance is known or unknown. In addition, its clinical significance needs
to be explained.
・ Secondary findings of potential germline variants: These should be included separately in thereport to warn treating physicians of potential clinical relevance.
・ Indicate in the report that the analysis does not find everything. Due to the limitations of theanalysis, the areas that the analysis could not cover well and where the sensitivity was low should
be included in the report.
Validation
・ Because the performance characteristics are different for each variant, it is necessary to verify theperformance (e.g. sensitivity) and perform a validation based on it.
・ Along with a demonstration of the quality sequence of all target areas without sample type bias,
the performance characteristics of each sample type (e.g. FFPE, FF, WB, BM, FNA) need to beestablished and verified.
・ Minimal data needed to establish important performance characteristics (prevalence, i.e., total
number of variants detected in all studies, normal and variant readings, tables, and graphs(histograms) of reading depth for each target area are recommended.)① Accuracy: At least three well-characterized reference samples (e.g. HapMap DNA NA12878,
NA19240, or genomes in a bottle) should be sequenced to determine robust laboratoryspecific error rates across all target regions (specificity). This error rate is expected to be < 2%.
② Initial validation: At least 50 patient samples shall be included, including all intended andtumor-type samples. However, if FFPE is included, at least 75% of the samples should bederived from FFPE to ensure that the assay is robust when utilizing the most degraded sample
input sources. These samples contain a representative distribution of reportable variants
across all target regions (including GC-rich sequences) and shall be verified by an independent
reference method. Independent reference methods cannot utilize the same technology as the
NGS platform unless they are implemented in different laboratories.
③ Complete validation: Ten positive samples shall be sequenced and verified for each type ofintended variant in each target region. The reported variants of clinical significance identified
during clinical testing for which ongoing validation has not yet been fully validated, that is,confirmation by an independent reference method, should be verified using at least 10
positive samples per intended type by an independent reference method.
④ Accuracy (in progress): At least three positive patient samples containing variants close to theassay’s specified sensitivity should be analyzed in three lines in the same manner using different
ISO 15189 Application Guidance Document for Molecular-Genetic Examination37

barcodes.
⑤ Reproducibility (between runs): For each type of variant, at least three positive patient
samples containing variants close to the assay’s specified sensitivity shall be analyzed by two
different technicians, if possible, three times separately using different barcodes (from original
DNA to sequencing and data analysis) on different days.
⑥ Analytical sensitivity: The analytical sensitivity of the assay for each type of variants shall beestablished. This can be initially established with a defined mixture of cell line DNA (not aplasmid), but should be verified with samples from 3–5 patients.
⑦ When multiplexing the sample with different barcodes, it should be ensured that there is nocrosstalk between the samples and the barcodes, and that the running patient/barcode
combination provides reproducible results for all target areas and variant types regardless ofthe patient/barcode.
・ The accuracy and reproducibility of the bioinformatics process should be verified for both thedetection of all types of variants and the identification and classification of individually barcodedand multiplexed patient samples, if applicable.
・ Initial validation should be performed using a single version of all analytical software.
c)standard methods used outside their intended scope.

・ The laboratory shall perform validation to ensure that the methods specified in the standard foruse outside the intended scope and the extensions and modifications to the methods specified in
the standard are suitable for their (new) intended use.
d)validated methods subsequently modified.

・ Validation should be performed based on scientific evidence and clinical usefulness. Scientificevidence is a validation record.
・Quality metrics and continuous quality control for validation.

① Core quality metrics: quality and quantity of nucleic acids
1)

Validation parameters
The requirements for DNA quality and quantity differ depending on the tissue source,
extraction method, and NGS measurement method.
The laboratory shall establish the following:(1) Minimum criteria to ensure accurate variant detection and reproducible results, dependingon the measurement requirements and established sensitivity.
(2) Standard procedures for adjusting DNA input depending on sample quality.

2)

Continuous QC
Therefore, a plan for ongoing monitoring should be established. Any changes in the extraction
protocols should be followed by close monitoring of all downstream processes to ensure
adequate measurement performance.

② Core quality metrics: library qualification and quantification
38

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

1)

Validation parameters
The laboratory shall standardize protocols for library qualification and quantification.
As a recommendation for library quantification, each laboratory shall validate a method thatis suitable for its needs.

2)

Continuous QC
The fragment sizes of the library shall be measured to ensure that they fall within the expected
narrow molecular weight range.
Continuous methods should be used to minimize primer dimers, adapter dimers, and broader
bands of higher molecular weights.

③ Core quality metrics: Depth of coverage
1)

Validation parameters
Requirements vary depending on the platform used and the application.
Coverage is defined as achieving adequate sensitivity and specificity in the regions of interest.
The laboratory shall establish the minimum criteria for the depth of coverage characteristic ofa particular region under standard assay conditions (coverage threshold).

2)

Continuous QC
Methods should be used to monitor the overall coverage and region coverage in each run. If
the coverage thresholds are outside the validated range, the sample should be subjected toreanalysis. If only local regions are affected, testing of that region can be performed using analternate method.

④ Core quality metrics: uniformity of coverage
1)

Validation parameters
The required level of coverage across the targeted regions shall be defined during the validation
stage.

2)

Continuous QC
The uniformity of coverage shall be monitored and compared to the levels established during
validation. If the coverage uniformity profile falls outside the expected profile as establishedby the validation, this can be indicative of errors during the testing process.

⑤ Core quality metrics: GC bias
1)

Validation parameters
GC content affects the sequencing efficiency and uniformity of coverage of the targeted
regions. The extent of GC bias in all parts of the genome included in the assay should bedetermined during validation.

2)

Continuous QC
The GC bias should be monitored in every run to detect changes in measurement performanceor sample quality issues.

⑥ Core quality metrics: cluster density and alignment rate
ISO 15189 Application Guidance Document for Molecular-Genetic Examination

39

1)

Validation parameters
The laboratory shall define the right balance between overclustering and underclustering, andoutline the steps to prevent and resolve both instances. As a general guideline, the percentage
of clusters passing the filter should be > 80%, and the alignment rate should be > 95%.

2)

Continuous QC
Cluster density and alignment rate shall be monitored in every run.

⑦ Core quality metrics: transition/transversion ratio
1)

Validation parameters
An important parameter for whole-genome or whole-exome sequencing is not required fortargeted sequencing. The transition/transversion ratio shall be comparable to publishedvalues.

2)

Continuous QC
The transition/transversion ratio shall be monitored for every sample of each run to evaluate
the performance. Ratios lower or higher than expected can indicate that the quality of thebase calls was low.

⑧ Core Quality Metrics: base call quality scores
1)

Validation parameters
The laboratory shall establish acceptable raw base call quality score thresholds for the assay
during validation. Preprocessing methods to remove low-quality base calls should beestablished to reduce the false-positive rate.

2)

Continuous QC
Quality scores and the quality of the signal/noise ratio shall be monitored in every run. Lowquality scores can lead to increased false-positive variant calls; thus, the results should beinterpreted with caution, and repeat tests can be indicated.

⑨ Core quality metrics: Mapping quality
1)

Validation parameters
Parameters for mapping quality should be established during validation, and should
demonstrate that the test only analyzes reads that map to the regions targeted by the assay.
Steps should be established to filter reads that map to non-targeted regions.

2)

Continuous QC
The proportion of reads that do not map to target regions shall be monitored in each run.
Poor mapping quality can be a result of non-specific amplification, capture of off-target
DNA, or contamination.

⑩ Core quality metrics: duplication rate.
1)

Validation parameters
The acceptable parameters for the maximum duplication rate should be established for eachassay. Filtering of duplicate reads by the analysis pipeline should be established to increase the40

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

amount of usable sequencing data and prevent skewing of allelic fractions.
2)

Continuous QC
The duplication rate should be monitored in every run and independently for each sample tomonitor library diversity.

⑪ Core quality metrics: Strand bias
1)

Validation parameters
Strand bias occurs when the genotype inferred from the information presented by the forward
and reverse strands disagrees. The laboratory shall define the tolerance level for strand bias
and determine specific criteria for the adoption of alternate testing.

2)

Continuous QC
Closeness of strand bias shall be monitored in all samples.

【Note】
・ Accuracy, precision, analytical sensitivity, analytical specificity, reportable range, reference range,
reference interval, or alternatives shall be measured to clarify analytical/clinical validity and clinicalusefulness.
・ The performance characteristics of the testing procedure obtained by the validated method shallmeet the customer’s needs in the evaluation for the intended use.
・ When conducting testing, it is necessary to clarify the scientific basis, such as analytical validity,
clinical validity, and clinical usefulness, of the testing.
・ Since molecular examination, especially gene panel testing, is often implemented as a laboratory
developed test (LDT), both the validity of the entire testing and the performance characteristicsof the testing procedure need to be evaluated and documented, including the entire testingprocedure.
【Terminology】
Accuracy:
・ Closeness of agreement between a measured quantity value and the true quantity value of ameasurand
・ The definition in NGS is the closeness of agreement between the nucleic acid sequence derived
from the assay and a reference sequence.
Precision:・ Closeness of agreement between indications or measured quantity values obtained by replicate
measurements on the same or similar objects under specified conditions. Measurement precision
is usually expressed as a measure of imprecision; that is, it is calculated as the standard deviationof the measurement.
・ The definition in NGS is the closeness to which repeated sequence analyses give the same
repeatability and reproducibility.
Repeatability:
ISO 15189 Application Guidance Document for Molecular-Genetic Examination41

・ To measure all operations of the analytical method multiple times in a short time under the same
conditions using the quality control material specified in 5.6.2.2.
Reproducibility:
・ To measure all operations of the analytical method multiple times by changing the measurement
conditions between different facilities.
Analytical sensitivity:
・ Sensitivity a) performance of the analytical method or instrument represented by the lower limit
of detection of a certain quantity value in measurement, b) performance of the analytical method
represented by the slope of the calibration curve. The minimum or change quantity value that the
instrument can detect or the percentage of response to changes in the measurand.
・ The definition for NGS is the expectation of detecting the targeted sequence variations by analysis,
if present.
Analytical specificity:
・ Ability to clearly distinguish the measurement target components that are expected to exist in
various components.
・ NGS is defined as the probability that sequence variation will not be detected by analysis whennone is present (the false positive rate is a useful measured value for sequencing assays).
Reportable range:
・ NGS is defined as the region of the genome in which a sequence of acceptable quality can bedetected by the testing.
Reference range:
・ The definition of NGS is the region of sequence variations that can be detected in an unaffected
population.
Robustness:・ Confirmation by measurement that the ability of the analytical procedure is not affected, even if
there are fluctuations in analytical method parameters.
Analytical validity:
・ The definition in genetic testing is that the testing method is established and quality control isproperly performed, such as obtaining highly reproducible results. It is evaluated based on
information such as the positive rate when there is a mutation, the negative rate when there is no
mutation, the existence of a quality control program, and the confirmatory testing methods.
Clinical validity:
・ The definition of genetic testing is that the testing results are well defined. It is evaluated based on
information such as the sensitivity (the positive rate with diseases), specificity (the negative rate
without disease), disease prevalence, positive predictive rate, negative predictive rate, and therelationship between genetic type and phenotype.
Clinical usefulness:
42

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

・ The definition for genetic testing is that there are clinical merits, such as the ability to obtain
information on future prospects or to link it to appropriate preventive and therapeutic methods,
to diagnosing the disease being tested. It is evaluated based on information such as the impact of
the testing results on the subject and the existence of effective countermeasures.
・ The laboratory shall have written information about the analytical validity, clinical validity, andclinical usefulness of the performed tests.
・ As method development proceeds, regular reviews should be carried out to verify that the needs ofthe customer are still being fulfilled. Any change in requirements requiring modifications to thedevelopment plan should be approved and authorized.
5.5.3 Documentation of Examination Procedures【NGS Wet Bench Process Documentation】
The laboratory shall use a documented standard operating procedure (SOP) of the analytic wet
bench process used to generate the NGS data.
① The detailed documentation of wet bench processes is a critical part of quality assessment inmedical laboratories.
② All standard operating protocols of DNA/RNA sample preparation, fragmentation, library
preparation, barcoding (molecular indexing), sample (specimen) pooling, and sequence
generation shall be documented to track each step and subsequent manipulations.
③ Medical laboratories that handle different types of samples (e.g. blood, formalin-fixedparaffin-embedded specimens) should develop standard operating procedures (SOPs) for
each validated sample (specimen) type.
④ Commonly used metrics include the percentage of reads mapped to the target region, thefraction of bases meeting specified quality and coverage thresholds, and average coverage/baseand target region.
⑤ The laboratory shall define and document acceptance and rejection criteria for the wet bench
process, including sample preparation and sequencing.
⑥ It is critical to determine and summarize regions that failed analysis (e.g. due to inadequate
coverage) if they are not covered by orthogonal technologies (such as Sanger sequencing).
【NGS Bioinformatics Pipeline Documentation】
The laboratory shall use a documented SOP for the bioinformatics pipeline used to analyze,
interpret, and report the NGS results.
① The laboratory shall document all algorithms, software, and databases (referred to ascomponents) used in the analysis, interpretation, and reporting of NGS results.
② The versions of each of these components in the overall bioinformatics pipeline shall berecorded and traceable for each patient result (Version Control).
③ For each component, the laboratory can use a baseline, default installation, or can customize
ISO 15189 Application Guidance Document for Molecular-Genetic Examination

43

the pipeline by using alternate configuration parameters in deploying individual bioinformatics
tools or in running specific algorithms.
④ In either case, the laboratory shall document any customizations that vary from default
configuration, namely which parameters, cutoffs, and values are used.
⑤ Most NGS bioinformatics analyses are conducted by aligning sequence reads to a reference
sequence. The reference sequence version number and assembly details shall also be identified.
⑥ When describing the bioinformatics pipeline, the laboratory should document the overall
workflow of the data analysis and include the input and output files for each process step.
⑦ For each step, the laboratory should develop and document quality control parameters for
optimal performance. For example, a laboratory shall determine acceptable criteria, such as
the number of reads passing instrument-specific quality filters in the primary step.
⑧ Criteria for variant calling are essential and called parameters include thresholds for readcoverage depth, variant quality scores, and allelic read percentages. Each of these requirements
applies to multigene panel applications, as well as exome and genome sequencing.
⑨ The laboratory should also document the bioinformatics processes that are used for reducing
a large variant data set to a list of causal relation and/or candidate genes and/or variants. For
example, in inherited disease assays, the laboratory should document approaches used to
identify recessive (latent), dominant (overt), and new variants.
⑩ Evidence of compliance for this requirement should be demonstration of appropriate
documentation and that the laboratory follows its outlined procedures.
【Note】
・ Variant calling shall be named according to HGVS nomenclature, allowing explicit mapping tostandardized reference IDs.
・ A complete procedure manual shall be available on the workbench or in the work area.
5.6 Ensuring Quality of Examination Results
5.6.1 General【NGS Wet Bench Process—Quality Management Program】
・ The quality assurance program follows the workflow path. The programs should assess the preexamination process of NGS, examination processes, and post-examination processes used insequence analysis through reporting.
・ The NGS quality program should be integrated within the institution’s overall quality assurance
program.
・ The overall goal of the quality program is to ensure that testing is clinically relevant. This isparticularly important for tests such as NGS, for which no comparative analytical results of greater
sensitivity can exist. The appropriateness of test orders and analytic decisions should be groundedin medical science and evidence.
44

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

【NGS Bioinformatics Pipeline—Quality Management Program】
・ The laboratory shall maintain a record of deviations from expected results, and document the root
cause investigative measures and the corrective action taken.
・ Evidence of compliance includes documentation of monitoring quality control metrics as well as
records describing any divergences, including appropriate investigative measures and subsequentcorrective actions.
・ Selection of biopsy specimens in the preanalytic process is important for the success or failure of
NGS-based testing. The appropriate requirements shall be set according to the platform being
used.
【Tissue processing and storage methods】
FFPE tissue:
・ This is a standard for pathological evaluation and can be stored for a long time.
・ There are concerns about nucleic acid fragmentation due to formalin fixation.
・ Tumor fractions of unstained FFPE sections can be evaluated from H&E-stained sections.
Frozen tissue:
・ There is little concern regarding nucleic acid fragmentation.
・ The presence of tumor tissue and the tumor fraction cannot be accurately evaluated.
・ Can be stored for a long time at −80 °C.
【Amount of tissue and percentage of tumor cells】
Amount of tissue:
・ If sufficient tissue is obtained, various NGS library preparation methods can be applied.
・ If only fine tissue is obtained, only the NGS library preparation methods that require a small
amount of nucleic acid can be applied.
Percentage of tumor cells:
・ If the proportion of tumor cells is high, various NGS library preparation methods can be applied.
・ If the proportion of tumor cells is low, more unstained sections are needed for nucleic acidextraction.
【Tissue collection methods】
Surgical biopsy:
・ A sufficient amount of tumor tissue is often obtained.
Biopsy with core needle:・ Only a limited amount of tumor tissue will be obtained.
【Ratio of surviving tumor cells】
Surviving cell tissue:
・ Ideal for pathological evaluation and NGS library preparation by PCR
Necrotic cell tissue:
・ Not suitable for NGS library preparation by PCR
ISO 15189 Application Guidance Document for Molecular-Genetic Examination45

【Amount of nucleic acid harvested and required amount】
Amount of harvest:
・ Depends on the amount of tissue and the proportion of tumor cells
Required amount:
Depends on NGS library preparation method【Note 1】For the handling of FFPE specimens, the “Guidelines on the handling of pathological tissue
samples for genomic medical treatment” can be referred to.
【Note 2】
It is necessary to set appropriate criteria for cytological specimens.
Analyzing FASTQ file and BAM file information, which are digital data obtained from NGS
analysis, is a dry-lab process, but it is the basis for ensuring the quality and accuracy of proper
execution of the wet-lab process. Therefore, the following items need to be referred to, theappropriate criteria set in advance according to the platform being used, and a check made as towhether the wet-lab process meets the criteria.
Depth of coverage:
Depth of coverage characteristics of a particular region under standard examination conditions
shall be confirmed. It is critical to define adequate coverage to achieve the proper sensitivity andspecificity in the region(s) of interest. When the coverage threshold is outside the specified range,
the region should be subjected to analysis by an alternate method (e.g. Sanger sequencing) or
require additional evaluation.
Uniformity of coverage:
The coverage across the targeted regions should be established to produce reliable sequencing
results. The uniformity of coverage (targeted regions) should be monitored and compared to theestablished values.
GC bias:GC bias affects the efficiency of sequencing reactions and uniformity of coverage of the targeted
regions. The amount of GC bias in all parts of the genome included in the assay should bedetermined during verification. GC bias should be monitored in every run to detect changes intest performance.
Transition/transversion ratio (Ti/Tv ratio):
The ratio of transitions to transversions (Ti/Tv) should be comparable to published values. The Ti/
Tv ratio should be monitored in every run to detect changes in test performance.
Base call quality scores:
An acceptable raw-base-call quality-score threshold should be established during validation.
Informatics filters should be established to eliminate any reads with raw base calls lower than theestablished quality score (for a Q score of 10, the probability that the base is incorrect is 10%, 1%
46

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

for a Q score of 20, and 0.1% for a Q score of 30). In long-read technologies, when the detectionof larger indels is of interest, alignments can tolerate lower base call quality because the sequencelength and accuracy at the base level are less critical. The quality of the signal-to-noise ratio should
be monitored by examining the quality scores and quality of the signal-to-noise ratio across a readfor each run. Quality scores among the existing instruments are not readily comparable from one
to another.
Mapping quality:
Mapping quality is an indicator of the uncertainty that a read is properly mapped to the genomic
position. During the validation, it should be demonstrated that the test analyzes only the reads
that map only to specific target regions. Informatics filter programs should be established toeliminate any reads that map to non-target regions and remove duplicate reads. The proportion ofreads that did not map to the target regions should be monitored during each run. When reads do
not match the reference sequence, this is an indication that the sample does not perform within
normal parameters, and those reads should be excluded from analysis. For applications that involve
enrichment steps, poor mapping quality can be a result of non-specific amplification, capture of
non-target DNA, or contamination.
Duplicate read success rate and removal of duplicate reads:
Informatics filter programs should be established to eliminate duplicate reads resulting from clonal
amplification (all but one with the highest quality score) during alignment. This should bemonitored to prevent skewing of allelic fractions.
First-base read success:Some platforms allow the early intra-assay evaluation of sequence reads to determine quality scoresand the number of reads that pass the established quality filters. The number of reads that passed
the established quality filters should be established during assay validation. Evaluation of quality
scores and the number of reads that pass the established quality filters early in the sequencing
process can be used to monitor for contamination, confirm proper sample loading, and ultimately
assess the likelihood of a successful run. Some platforms allow a run to be prematurely terminatedif the established quality parameters are not met.
Decline in signal intensity:
During assay validation, the expected signal intensity across a read should be evaluated to establish
normal measurement performance ranges and the expected decline in signal intensity. (Signal
intensity across the read length depends on the platform.) Signal intensity attenuation should bemonitored on a run-by-run basis. A sudden reduction or increase in signal intensity indicates anerror in sequencing chemistry.
Strand bias:
Bias between DNA strands occurs when the data obtained from the forward and reverse strands
are different. It is necessary to check the degree of bias that is acceptable to the results. Interchain
ISO 15189 Application Guidance Document for Molecular-Genetic Examination47

bias should be monitored for all samples.
5.6.2 Quality Control
5.6.2.1 General・ The testing facility shall establish policies and procedures for regularly evaluating quality assurance
in the facility, and document the evaluation results and measures taken for improvement.
・ Routine application of validated bioinformatics pipelines should be accompanied by monitoring
laboratory-determined quality control metrics.
・ Divergence from expected quality metrics during the analysis of clinical samples (specimens)
requires investigation and resolution.
【Note】
・ It is necessary to monitor the quality indicator to ensure that the analysis is always performed
correctly. Sampling and nucleic acid extraction are also important factors in all molecularexamination, so regular monitoring is needed with proper quality control materials close to thespecimens.
・ Quality control procedures shall be implemented to monitor the performance of the examination
process. Control procedures are designed to detect test system failures, environmental disadvantages,
and immediate errors due to operator performance, and to monitor test performance accuracy andprecision over time. Although sequence analysis is usually considered a qualitative analysis, NGS
has both qualitative and quantitative aspects to consider when devising effective control andcontrol procedures.
・ NGS quality control materials and metrics should be established during validation. QC materials
and metrics should be established for each component of the NGS testing process, including
DNA extraction, library preparation, DNA sequencing, and informatics analysis pipelines. The
laboratory shall use appropriate quality control procedures to ensure all aspects of the sequencing
process, including instrument sample performance, base calls, alignments, and variant calls.
5.6.2.2 Quality Control Materials
・ A combination of multiple quality control materials should be used as much as possible. It isnecessary to regularly use quality control materials that are the reference standards or traceable toreference standards, not limited to in-kit control.
・ The level and type of quality control depends on the importance, content of the analysis, frequencyof the analysis, size of the batch, degree of automation, and difficulty and reliability of the testing.
・ As the nucleic acid amplification test is highly sensitive, it is necessary to pay attention to crosscontamination. Negative controls shall be used. Parallel runs are desirable for negative controls,
but in the case of instruments for single assays, it is necessary to avoid the risk of cross-contaminationby using alternatives.
48

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

・ To ensure a negative result, it is necessary to ensure that the nucleic acid amplification reaction is
performed correctly using an internal control.
・ For regular quantitative tests, at least two concentrations of positive and negative controls shall be
used. For qualitative tests, one concentration of positive and negative controls shall be used. In
either case, it is necessary to establish and monitor quality control procedures, including nucleic
acid extraction steps. Quality control materials that can closely examine the steps of nucleic acidextraction that are close to the specimens should be used. Internal positive controls should also be
regularly used.
・ Negative controls shall be added to every amplification step to ensure that there was nocontamination between the samples (specimens) and the reagents. This control can be analyzed in
an appropriate way before actual sequencing.
・ It is necessary to verify the accuracy and specificity of the analysis to detect variants, includingreference genomes recommended by NIST or equivalent institutions, such as NA12878 and/or
GM24385 and/or NA19240, as negative controls during and thereafter on a regular basis.
・ Positive/sensitivity controls shall be included in each run. It is suggested that these controls be
individually barcoded, low-positive DNA samples containing multiple known variants of eachtype that were detected near analytical sensitivity to verify that a low percentage of variants is
consistently identified. If not whole variants, and if all target areas can be included in a single
control sample, then a defined rotation schedule should be used.
【Note】
・ Although it is a “standard material” in the literature, standard materials have not been developed
for many tests. Therefore, it is paraphrased as “reference standard”
・ Products approved by pharmaceutical affairs are marketed by guaranteeing negative results without
the use of internal controls.
・ The level of selected quality controls shall be sufficient to ensure the validity of the results. Various
quality controls can be used to monitor various fluctuations during the process. By analyzing thequality control materials for each batch of samples, the fluctuation tendency of the system can beunderstood. The use of various blanks reveals the contribution to the risk, as well as the contribution
from the components being analyzed.
5.6.2.3 Quality Control Data
・ Internal quality control takes various forms, including blanks, reference standards, spiked samples,blind materials, iterative analysis, and the use of quality control materials. In particular, X-R
control charts, twin plots, and multi-rule control methods should be used for monitoring withquality control materials.

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

49

5.6.3 Interlaboratory Comparisons
5.6.3.1 Participation【Note 1】・ Interlaboratory comparison means “organization, performance, and evaluation of measurementsor tests on the same or similar items by two or more laboratories in accordance with predetermined
conditions.”
・ The proficiency test refers to the “evaluation of the performance of participating laboratories
against a predetermined standard by comparison between laboratories.”【Note 2】・ International large-scale external accuracy evaluation programs for molecular-genetic tests include
CAP (College of American Pathologists) and GenQA/EMQN (European Molecular Genetics
Quality Network).
・ The facility proficiency testing system should be structured to evaluate all stages of the examination
process performed in the laboratory, including reporting results.
5.6.3.2 Alternative approach・ If a facility proficiency testing system is not available, testing facilities should implement alternative
methods.
・ Alternative methods include exchanging samples among laboratories, repeating tests of samples,
conducting tests by different independent methods, and comparison with other parameters. Suchalternative methods can also include multiple proficiency tests that examine individual steps in theanalytical process (e.g. sequence).
【Note】
・ Note that alternative approaches are different for molecular-genetic examination.
・ Measured values can differ between facilities; therefore, this point should be taken into considerationin the evaluation method.
・ Currently, external quality assesment surveys for molecular and cytogenetic tests established bydomestic institutions are a qualitative test for the tubercle bacillus complex, and quantitative tests
for hepatitis B virus and hepatitis C virus provided by the Japanese Association of Medical
Technologists.
5.7 Post-examination processes
5.7.2 Storage, Retention, and Disposal of Clinical Samples
・ The biobank has a policy for handling submitted clinical specimens: ① Purpose of intended useof specimen, ② Required specimen data, ③ Safety procedures considering the types of clinicalspecimens/pathogen and required biosafety/risk level, ④ Duration of storage.
・ Procedures include the duration of sample storage, the control method of remaining samples,50

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

disposal method of samples, possibility of being re-contacted when sample retests are required(e.g. due to significant advances in knowledge or technology), the possibility of secondary use for
quality control under anonymity, the possibility of access to the samples by a third party, and themethod of confidentiality protection (symbolization/anonymization).
・ It is also necessary to coordinate the handling of the sample after the test in advance with theparties concerned.
・ The administrator is responsible for discarding the tested samples after a specified period of storage
to prevent unintended use.
・ The sample processing standard operating procedure includes the following items: It is necessary
to clarify the acceptable criteria for handling the return, retest, and additional tests of the sample(hereinafter referred to as “return, etc.”) from the referring customers by setting standards forstorage, return, and disposal of each sample and standardizing these operations.
① Regarding the setting of the storage period for each sample, it should be set appropriately
based on the nature of the sample and the frequency of return and display the storage
conditions such as room temperature, refrigeration, and freezing. In addition, it is desirable
that a specimen for which an emergency report has been made should be stored for a certain
period of time after the report has been submitted to the referring customer.
② It is necessary to describe the confirmation items and return procedure when the referring
customer requests a return, the confirmation items before disposal of the sample whose
storage period has expired, and disposal procedure.
・ The period of sample storage shall be set considering the characteristics of the sample (specimen)
and the appropriate storage environment.
・ DNA quality declines over time
・ Biobanks shall store the appropriate volume of sample, as freeze/thaw cycles for the sample(specimen) can be deleterious to sample quality.

【Note】・ For example, properly stored FFPE specimens can be used within five years.
・ The use, storage, transfer, and disposal of samples (specimens) taken for molecular examination
shall be in accordance with legal, ethical, and professional standards.
5.8 Reporting of Results
5.8.1 General・ The report shall be able to convey the information properly, considering that the recipient can not
be a professional healthcare personnel. (This is common to ① nucleic acid tests for pathogens, ②molecular-genetic tests for human somatic gene alterations, and ③ human genetic testing, but, asa general rule, mainly applies to ③ human genetic testing.)
・ The report should be accurate, concise, and comprehensive, and should include all essential
ISO 15189 Application Guidance Document for Molecular-Genetic Examination51

information so that the subject experts can make appropriate decisions.
【Reporting of Scondary Genetic Findings】
The laboratory has a policy for reporting secondary genetic findings unrelated to the clinicalpurpose of testing.
① Clinically significant genetic findings that are unrelated to the phenotype for testing canoccur when performing single gene, gene panel, exome, and whole genome sequencing.
② Restricted sequence analysis of a panel of genes that are relevant to the diagnosis of a particulardisease state (either with targeted sequencing or targeted bioinformatics analysis) can limit,
but not eliminate, the potential for secondary findings. This can include identification ofvariants relevant to autosomal dominant (overt) diseases, carrier status for recessive (latent)diseases, predisposition to adult-onset dominant (overt) conditions (including cancer andneurodegenerative conditions), and drug response alleles commonly known as pharmacogenetic
markers.
③ The laboratory starting to use NGS for clinical testing should be aware of the potential forfinding incidental, clinically significant results and should have a policy in place for whether
these results will be reported for those assays where such incidental findings are expected (e.g.
exome).
④ The recently published ACMG recommendations for reporting medically actionable
incidental findings include a minimum gene list for which, if a known mutation is found, itshould be reported. Laboratories can choose to follow the ACMG recommendations but are
not necessarily expected to report only the findings of these genes.
⑤ Laboratories can also develop their own policies regarding the return of incidental results. If
the laboratory’s policy is not to report incidental findings or to limit reporting to a subset ofvariants related to a particular disease state, this should be clearly stated in the laboratory
report for assays where incidental findings are expected.
⑥ Ethical considerations should also be taken into account when deciding whether to reveal
certain genetic information to patients.
⑦ The level of risk associated with disclosing incidental findings depends on the severity of thedisease, clinical actionability, and other risk-benefit indicators. For example, common diseaserisk alleles, such as for type 2 diabetes or cardiovascular disease, which have a small effect size
(low relative risks), or pharmacogenetic risk information, can have different consequences
than genetic information indicating a predisposition to cancer or a Mendelian disorder thatcan or cannot be medically treatable. All these aspects should be considered before returning
the results to patients.
【Note】It should be desirable to refer to the “Proposals concerning the information transmission process
in genomic medicine-regarding comprehensive tumor genomic panel tests and germline whole52

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

genome / whole exome analysis” in the description of the secondary genetic findings report.
5.8.3 Report Content
a)a clear, unambiguous identification of the examination including, where appropriate, theexamination procedure.
The report should use internationally accepted terminology and nomenclature, such as using
standard methods for describing nucleotide sequences.

b)the identification of the laboratory that issued the report.

・ Name and address of testing performed facility
・ Contact information for the testing facilityc)identification of all examinations that have been performed by a referral laboratory.

・ When conducting testing at a referral facility, the name and location of the referral facilityd)patient identification and patient location on each page.

・ Subject identification informatione)name or other unique identifier of the requester and the requester’s contact details

・Name and contact information of medical doctors and/or other test requesters.
f ) date of primary sample collection (and time, when available and relevant to patient care).
・ Sample (specimen) collection date and receipt dateg) type of primary sample.
・ Sample (specimen) typei) examination results reported in SI units, units traceable to SI units, or other applicable units.
・ Testing results (when making an interim report of testing results, it is necessary to include thecontents in the final report)k) interpretation of results, where appropriate.
・ Information necessary for interpreting testing results (reports should endeavor to provide sufficient
information to requesters, including appropriate medical interpretations with clinical significance)
・ At the least, the reports should include the following information:① Subject identification information② Name of medical doctors and/or other test requesters and its contact information③ Reasons for applying the test and medical information of the subject required to perform thetesting.
④ Testing and methods performed (including scope of analysis, test limitations, and analysis
sensitivity and specificity)⑤ Specimen type
⑥ Specimen collection date and receipt date
⑦ Information on the condition of the specimen (additives, transportation, and storage
conditions, if necessary)
ISO 15189 Application Guidance Document for Molecular-Genetic Examination

53

⑧ Name and address of the laboratory where the specimen was actually tested (including thereferral laboratory if re-referred)⑨ Testing results (when making an interim report of testing results, it is necessary to reflect thecontents in the final report)⑩ Information necessary for interpreting testing results (reports should endeavor to provide
sufficient information to requesters, including appropriate medical interpretations withclinical significance)⑪ Affiliation, title, and name of the report author and final administrator⑫ Laboratory contact information⑬ Date of the report release【Note】
・ The information necessary for interpreting the results is the subject’s clinical data, pedigree
information, race, clinical sensitivity, and specificity of the specimen. The report shall be clearlystated so that the recipient can understand the clinical usefulness and limitations of the test results.
If the quantity and quality of the specimen received can affect the results, it shall be stated in thereport.
・ As a general rule, the following information should be included in the testing report: The main
subject of the report is genetic testing, but in some cases, nucleic acid tests for pathogens andmolecular-genetic tests for somatic gene alterations are also assumed.
① The need for genetic counseling by qualified genetic counseling professionals② Potential impact on family③ Information such as necessary additional testing・ All information regarding the interpretation of the results should be attached to the final report,
including when the testing was re-outsourced.
l)other comments such as cautionary or explanatory notes (e.g. quality or adequacy of the primary
sample which may have compromised the result, results/interpretations from referral laboratories,
use of developmental procedure).

・ Information on the condition of the sample (specimen) (e.g. additives, transportation, and storage
conditions, if necessary)n) identification of the person(s) reviewing the results and authorizing the release of the report (if notcontained in the report, readily available when needed).
・ Affiliation and name of the final administrator of the reporto) date of the report, and time of release (if not contained in the report, readily available when needed).
・ Date of the report release
5.9 Release of Results
5.9.1 General54

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

【Note1】・ Since genetic counseling is important not only for providing information but also for psychosocial
support that enables autonomous selection of patients and subjects, it is desirable that medical
doctors with abundant medical experience of the disease and persons who are proficient in genetic
counseling cooperate and carry out medical care as a team.
・ If necessary, medical doctors, medical doctors licensed as clinical geneticists, doctors licensed asclinical laboratory specialists, and persons in charge (such as pharmacists, nurses, and clinicallaboratory technicians) who have specialized knowledge about pharmacogenomics testing should
explain the results to the subjects (see 5.4.2).
【Note2】・ Genetic counseling includes the following:① Interpretation of family history and medical history to assess the occurrence and recurrenceof the disease② Education on genetic phenomena, testing, management, prevention, resources, and research③ Informed choice (autonomous choices after obtaining sufficient information) and counselingto facilitate adaptation to risks and situations【Note3】
Handling genetic information when using it for medical treatment:
・ Genetic information obtained by genetic testing includes information on the constitution andonset of illness due to changes in a person’s genes and chromosomes, as well as information on
relatives. Since this information does not change over a person’s life, if it leaks, it can cause serious
damage and suffering to the person and relatives. Therefore, regarding the handling of genetic
information obtained by genetic testing, it is necessary to pay particular attention to the guidelines
stipulated in the UNESCO International Declaration and the guidelines stipulated by relatedorganizations.
・ Even if a person agrees to carry out the testing, the person and the family members often havegreat anxiety, because it is difficult for them to understand the meaning of the test result accurately
and how to deal with the future predictability of the disease. Therefore, when a medical institution
conducts genetic testing, it is necessary to provide psychosocial support to the person and his/herfamily, such as genetic counseling by a person with expertise in clinical genetics.
5.10 Laboratory Information Management
5.10.1 General・ Computer access codes (security codes, user codes) shall be in place, and the security of the accesscodes shall be maintained (inactivated when the employees leave).
・ Personal genetic information shall comply with the Personal Information Protection Law andguidelines based on it (see 5.4.2).
ISO 15189 Application Guidance Document for Molecular-Genetic Examination55

・ Information security measures should comply with relevant laws and regulations such as the Act
on the Protection of Personal Information. In addition, the laboratory should refer to “Guidelines
for Safety Management of Medical Information Systems” (issued by the MHLW Health Policy
Bureau on March 31, 2005, No.0331009, PFSD Notification No. 0331020, Insurance Notification
No. 0331005 Director of Medical Affairs Bureau, Ministry of Health, Labor and Welfare Bureau,
Director of Pharmaceutical and Food Safety Bureau, Insurance Director Notification attached).
【NGS data transfer confidentiality policy】
The laboratory shall have a policy and procedures describing processes to ensure that patient
confidentiality and security are maintained during internal and external storage and transfer ofsequencing data.
① Next-generation sequencing generates significant amounts of data, particularly of genesequences that, with other information such as name, date of birth, medical record number,and other components of protected health information, can potentially be used to identify
individual patients.
② The laboratory shall establish rigorous processes to ensure the protection and privacy of thisinformation.
③ The laboratory shall have robust policies regarding the transfer of genomic information toother health care entities and third-party vendors, such as those providing cloud-based
computing resources or registered clinical laboratory services. Procedures to ensure
confidentiality include data encryption, secure data transfer, user authentication with
controlled access to protected health information, and audit trails that track the transmissionof data as well as the receiving entities and/or users.
④ The laboratory performing large-scale genomic sequencing analysis for clinical testing should
be aware of efforts to study the medical and ethical implications of returning incidental
results of NGS and consider these when developing their reporting policies.
・ Regarding personal information protection and information security, it shall clearly state its
compliance with the relevant laws and regulations, such as the Personal Information Protection
Law and the “Guidelines for Safety Management of Medical Information Systems” prepared by
the Ministry of Health, Labor and Welfare.
・ There shall be policies and procedures for protecting the confidentiality of personal information
obtained from samples (specimens) and testing results.
・ When referring to molecular-genetic tests, various safety management measures (such asorganizational, human, physical, and technical safety management measures) should be takenfrom the viewpoint of personal information protection, patient names shall be anonymized, andspecial attention should be paid to the handling of samples (specimens) and testing information
through all processes from sample (specimen) collection to reporting of test results.
・ In genetic tests aimed at diagnosing monogenic diseases, personal genetic information should be
56

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

protected by anonymization and confidential reports.
・ Regarding the handling of biological samples (specimens) after testing, the administrator is
responsible for discarding the biological samples after a clear storage period, even if they have beenused in other studies.
【Note 1】・ As a general rule, the results of genetic tests performed to diagnose patients who have alreadydeveloped a disease should be recorded in medical records as information shared by medical
professionals related to the diagnosis of patients, similar to the results of other clinical tests. The
genetic information of an individual obtained by genetic testing, such as all medical information,is subject to confidentiality and should not be disclosed to third parties, including relatives,
without the consent of the subject. With the consent of the subject, if it is considered that thediagnosis result of the subject is useful for the health management of the relatives and cannot leadto effective prevention or treatment without the information, consideration shall be given todisclosing it to the relatives. Genetic counseling is provided at the right time as needed for genetic
testing and diagnosis.
・ Genetic tests performed for the purpose of non-onset carrier diagnosis, presymptomatic diagnosis,
and prenatal diagnosis shall be performed after appropriate genetic counseling has been performedin advance. Genetic counseling helps people understand and adapt to the genetic, psychological,
and family impacts of the genetic involvement of a disease. This process includes ① family history
and medical history interpretation to assess the development and recurrence of the disease; ②
education on genetic phenomena, testing, management, prevention, resources, and research;③informed choice (autonomous choices after obtaining sufficient information); and ④ counselingto facilitate adaptation to risks and situations.
・ The subjects of genetic testing shall be divided into patients who had already developed the disease
and others. PGx is equivalent to patients who have already developed the disease. Pharmacology
genomics tests included in pharmacogenomics tests deal with genetic information of germline
lines, but unlike genetic information of monogenic diseases, in clinical practice, it can be treatedin the same way as normal medical information after referring to the related guidelines.
【Note 2】・ When conducting genetic testing, the medical doctor in charge shall obtain informed consent
regarding genetic testing from the subject in advance.
・ Samples for genetic testing shall be stored strictly in accordance with safeguards, and theconfidentiality of personal identification information and personal genetic information as test
results should be protected.
① As a general rule, general medical information and genetic information linked to a specificindividual should be stored separately.
② Personal identification information and individual genetic information are subject toISO 15189 Application Guidance Document for Molecular-Genetic Examination57

confidentiality obligations, and the medical doctor in charge, genetic counseling staff, andthe person in charge of the medical institution shall strictly protect and manage them toprevent leakage to third parties.
③ When a part of genetic testing is referred to another testing institution/facility, the sample
shall be anonymized in advance, and the confidentiality of personal identification information
shall be maintained.
【Note 3】・ Personal information is “information that can identify a specific individual by the name, date ofbirth, or other description contained in the information (including those that can be easily collatedwith other information and thereby identify a particular individual)” (JIS Q 15001)
・ ” Personal identification code” refers to characters, numbers, symbols, and other codes stipulated
by law as being able to identify a specific individual from the information alone, and informationthat includes such information is personal information. Among the characters, numbers, symbols,and other codes converted to use any of the physical characteristics listed in ① to ⑦ for use incomputers, “those that meet the standards stipulated by the rules of the Personal Information
Protection Commission as sufficient to identify a specific individual” are said to correspond to
personal identification codes.
① Concerning DNA, those pieces of information that authenticate the person by genotype
information such as whole-nucleus genome sequence data, whole exome sequence data,
whole-genome single nucleotide polymorphism (SNP) data, sequence data consisting of 40
or more independent SNPs, and a short tandem repeat (STR) of four bases above nine loci.
② Those that can authenticate the person using a device or software that aims to authenticate
the person with the characteristic information extracted from the skeleton and color of theface and the position and shape of the eyes, nose, mouth, and other parts of the face.
③ Those that can authenticate a person with a device or software that aims to authenticate theperson with the characteristic information extracted from the linear pattern formed by the
undulations on the surface of the iris using infrared light or visible light.
④ Those that can authenticate the person with a device or software that aims to authenticate theperson, such as a speaker recognition system, for characteristic information related to vocal
cord vibration, glottis opening/closing, vocal tract shape, and voice changes.
⑤ Those that can authenticate the person using a device or software that aims to authenticate
the person with characteristic information extracted from the posture, the movement of botharms, stride length, and other walking modes during walking.
⑥ Those that can authenticate the person using a device or software that aims to authenticate
the person with the characteristic information extracted from the shape of the vein determinedby the branch and the end point of the vein under the skin of the palm, back of the hand, or
finger using infrared light or visible light.
58

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

⑦ (fingerprint) Those that can authenticate the person using a device or software that aims toauthenticate the person with the characteristic information extracted from a fingerprint
formed by a ridge on the surface of a finger.
(palm print) Those that can authenticate the person using a device or software that aims toauthenticate the person with the characteristic information extracted from the palm print
formed by ridges and wrinkles on the surface of the palm.
⑧ Combination
Those that can authenticate the person by a device or software that aims to authenticate theperson by combining the characteristic information extracted from ① to ⑦.
・ “Personal information requiring special consideration” refers to information that requires special
consideration in handling so as not to cause unreasonable discrimination, prejudice, or otherdisadvantages. The information that corresponds to the personal information requiring special
consideration in medical institutions and care-related businesses is the medical history recorded in
medical records or care-related records, medical information and dispensing information, resultsof health examinations and contents of health guidance, facts of disabilities (such as physicaldisabilities, intellectual disabilities, mental disorders), and the facts of being harmed by crimes that
medical professionals could know about the patient’s physical condition, medical condition,
treatment in the process of medical treatment, and dispensing.
5.10.3 Information System Managementa)validated by the supplier and verified for functioning by the laboratory before introduction, withany changes to the system authorized, documented and verified before implementation.

・ Computer programs should be checked for proper performance after installation of new systems
or modifications of existing systems.
・ Any changes or modifications to the system should be documented and approved.
・ Documentation shall be retained for at least two years beyond the service life of the system in thebiobank.

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

59

6

Reference1)

ISO 15189, Medical laboratories - Requirements for quality and competence

2)

Aziz N, Zhao Q, Bry L, Driscoll DK, et al. College of American Pathologists’ Laboratory
Standards for Next-Generation Sequencing Clinical Tests, Arc Pathol Lab Med 2014; 1-13.

3)

Code of Federal Regulations 42 Part493, Laboratory Requirements, Subpart M - Personnel
for Nonwaived Testing. 1992.

4)

College of American Pathologists. Biorepository Accreditation Program Checklist. 2019;
1-64.

5)

ISO 15190, Medical laboratories - Requirements for safety.

6)

ISO 35001, Biorisk management for laboratories and other related organizations.

7)

ISO 20658, Medical laboratory examinations - Requirements for collection, transport,
receipt and handling of samples.

8)

Smith J, Johnson P, Jacoby R, et. al. Quality Management Systems - Process Validation
Guidance. GHTF/SG3/N99-10: 2004. 1-36.

9)

ISO/IEC Guide 99, International vocabulary of metrology - Basic and general concept andassociated terms (VIM).

10) Hardwick SA, Deveson IW, Mercer TR. Reference standards for next-generation sequencing,
Nat Rev Genet 2017; 18(8): 473-484.
11) ISO TS 20658, Medical laboratory examinations - Requirements for collection, transport,
receipt and handling of samples.
12) ISO 17822, In vitro diagnostic test systems – Quantitative nucleic acid based in vitro
examination procedure for detection and identification of microbial pathogens – Quality
practices for nucleic acid amplification.
13) ISO 20166-1, Molecular in vitro diagnostic examinations - Specifications for pre-examination
processes for formalin-fixed and paraffin - embedded (FFPE) tissue - Part 1: Isolated RNA.
14) ISO 20166-2, Molecular in-vitro diagnostic examinations - Specifications for pre-examination
processes for formalin-fixed and paraffin - embedded (FFPE) tissue - Part 2：isolated
proteins.
15) ISO 20166-3, Molecular in-vitro diagnostic examinations - Specifications for pre-examination
processes for formalin-fixed and paraffin-embedded (FFPE) tissue - Part 3：isolated DNA.
16) ISO 20184-1, Molecular in vitro diagnostic examinations - Specifications for pre-examination
processes for frozen tissue - Part 1: Isolated RNA.
17) ISO 20184-2, Molecular in-vitro diagnostic examinations - Specifications for pre-examination
processes for frozen tissue – Part 2: Isolated proteins.
18) ISO 20186-3, Molecular in vitro diagnostic examinations - Specifications for pre-examination
processes for frozen tissue - Part 3: Isolated DNA.
60

ISO 15189 Application Guidance Document for Molecular-Genetic Examination19) ISO 21474-1, In vitro diagnostic medical devices - Multiplex molecular testing for nucleic
acid - Part 1: Terminology and general requirements for nucleic acid quality evaluation.
20) ISO 21474-1, In vitro diagnostic medical devices - Multiplex molecular testing for nucleic
acid - Part 1: Terminology and general requirements for nucleic acid quality evaluation.
21) ISO 20387, Biotechnology-Biobanking - General requirements for biobanking.
22) Jennings LJ, Arcila ME, Corless C, et al. Guidelines for Validation of next - generation
sequencing - Based oncology panels, J Mol Diagn 2017, 19(3), 341-365
23) ISO/IEC 17025, General requirements for the competence of testing and calibrationlaboratories.
24) Gargis AS, Kalman L, Berry MW, et al. Assuring the quality of next-generation sequencing
in clinical laboratory practice, Nat Biotechnol 2021; 30(11): 1033-1036.
25) Roy S, Coldren C, Karunamurthy A, et al. Standards and Guidelines for validating nextgeneration sequencing bioinformatics pipelines, J Mol Diagn 2018; 20(1): 4-27.
26) Zucker HA, Dreslin S. “Next Generation” Sequencing (NGS) guidelines for somatic genetic
variant detection, New York State of Opportunity. 2018; Mar. 1-6.

ISO 15189 Application Guidance Document for Molecular-Genetic Examination

61