February 2014

MM09-A2Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine; Approved Guideline—Second Edition

This document addresses diagnostic sequencing using both automated capillary-based sequencers and massively parallel sequencing instruments. Topics include specimen collection and handling; isolation and extraction of nucleic acid; template preparation; sequence generation, alignment, and assembly; validation and verification; ongoing quality assurance; and reporting results.

A guideline for global application developed through the Clinical and Laboratory Standards Institute consensus process.

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Clinical and Laboratory Standards Institute Setting the standard for quality in clinical laboratory testing around the world.

The Clinical and Laboratory Standards Institute (CLSI) is a not-for-profit membership organization that brings together the varied perspectives and expertise of the worldwide laboratory community for the advancement of a common cause: to foster excellence in laboratory medicine by developing and implementing clinical laboratory standards and guidelines that help laboratories fulfill their responsibilities with efficiency, effectiveness, and global applicability. Consensus Process

Consensus—the substantial agreement by materially affected, competent, and interested parties—is core to the development of all CLSI documents. It does not always connote unanimous agreement, but does mean that the participants in the development of a consensus document have considered and resolved all relevant objections and accept the resulting agreement. Commenting on Documents

CLSI documents undergo periodic evaluation and modification to keep pace with advancements in technologies, procedures, methods, and protocols affecting the laboratory or health care.

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Comments on published CLSI documents are equally essential, and may be submitted by anyone, at any time, on any document. All comments are addressed according to the consensus process by a committee of experts. Appeals Process

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ISBN 1-56238-953-X (Print) MM09-A2

ISBN 1-56238-954-8 (Electronic) Vol. 34 No. 4

ISSN 1558-6502 (Print) Replaces MM09-A

ISSN 2162-2914 (Electronic) Vol. 24 No. 40

Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine;

Approved Guideline—Second Edition

Volume 34 Number 4

Thomas J. Lenk, PhD

Ira M. Lubin, PhD, FACMG

David Ross, PhD

Matthew J. Bankowski, PhD, D(ABMM)

Sarah F. Bennett, MT(ASCP)

Carey-Ann Burnham, PhD, D(ABMM)

Eric J. Duncavage, MD

Lisa M. Farwell

Tamara V. Feldblyum, MS

Manohar R. Furtado, PhD

Damon K. Getman, PhD

Tina M. Hambuch, PhD

Roger D. Klein, MD, JD, FCAP

Jamie L. Platt, PhD, CGMBS, MB(ASCP)

Lakshman Ramamurthy, PhD

Hans Scheffer, PhD

Tracy L. Stockley, PhD, FCCMG, FACMG

Patrik Vitazka, MD, PhD, FACMG

Emily S. Winn-Deen, PhD

Abstract Sequencing methods for genotyping have moved from the research laboratory into the clinical laboratory. Sequencing is an assay

format of choice for very high-complexity genotyping, especially when hundreds or thousands of bases of genetic sequence are

analyzed. Clinical and Laboratory Standards Institute document MM09-A2—Nucleic Acid Sequencing Methods in Diagnostic

Laboratory Medicine; Approved Guideline—Second Edition addresses diagnostic sequencing using both automated capillary

electrophoresis sequencers and massively parallel sequencing instruments. Topics covered include specimen collection and

handling; isolation and extraction of nucleic acid; template preparation; sequence generation, alignment, and assembly; validation

and verification; ongoing QA; and reporting results.

Clinical and Laboratory Standards Institute (CLSI). Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine;

Approved Guideline—Second Edition. CLSI document MM09-A2 (ISBN 1-56238-953-X [Print]; ISBN 1-56238-954-8

[Electronic]). Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087 USA,

2014.

The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through

two or more levels of review by the health care community, is an ongoing process. Users should expect revised editions of any

given document. Because rapid changes in technology may affect the procedures, methods, and protocols in a standard or

guideline, users should replace outdated editions with the current editions of CLSI documents. Current editions are listed in

the CLSI catalog and posted on our website at www.clsi.org. If you or your organization is not a member and would like to

become one, and to request a copy of the catalog, contact us at: Telephone: 610.688.0100; Fax: 610.688.0700; E-Mail: customerservice@clsi.org; Website: www.clsi.org.

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Copyright ©2014 Clinical and Laboratory Standards Institute. Except as stated below, any reproduction of

content from a CLSI copyrighted standard, guideline, companion product, or other material requires

express written consent from CLSI. All rights reserved. Interested parties may send permission requests to

permissions@clsi.org.

CLSI hereby grants permission to each individual member or purchaser to make a single reproduction of

this publication for use in its laboratory procedure manual at a single site. To request permission to use

this publication in any other manner, e-mail permissions@clsi.org.

Suggested Citation

CLSI. Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine; Approved Guideline—

Second Edition. CLSI document MM09-A2. Wayne, PA: Clinical and Laboratory Standards Institute;

2014.

Approved Guideline December 2004

Approved Guideline—Second Edition February 2014

ISBN 1-56238-953-X (Print)

ISBN 1-56238-954-8 (Electronic)

ISSN 1558-6502 (Print)

ISSN 2162-2914 (Electronic)

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Contents

Abstract .................................................................................................................................................... i

Committee Membership ........................................................................................................................ iii

Foreword .............................................................................................................................................. vii

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

2 Introduction ................................................................................................................................ 1

3 Standard Precautions .................................................................................................................. 2

4 Terminology ............................................................................................................................... 2

4.1 A Note on Terminology ................................................................................................ 2 4.2 Definitions .................................................................................................................... 2 4.3 Abbreviations and Acronyms ....................................................................................... 9

5 Technologies and Applications ................................................................................................ 11

5.1 General Technology Overview ................................................................................... 11 5.2 Applications Overview ............................................................................................... 18

6 Specimen Collection, Storage, Transport, and Preparation for Analysis ................................. 24

6.1 Specimen Collection ................................................................................................... 24 6.2 Specimen Transport and Storage ................................................................................ 25 6.3 Nucleic Acid Extraction and Purification ................................................................... 25 6.4 Sample Storage and Retention .................................................................................... 26

7 Template Preparation ............................................................................................................... 26

7.1 Primer Design and Synthesis for Polymerase Chain Reaction and Sequencing ......... 27 7.2 Amplification Parameters ........................................................................................... 29 7.3 Practices to Aid in Contamination Control ................................................................. 30 7.4 Polymerase Chain Reaction Product Purification ....................................................... 31 7.5 Quantity and Quality of DNA Template ..................................................................... 33 7.6 Library Construction for Massively Parallel Sequencing ........................................... 33 7.7 Unique Sample Preparation Considerations ............................................................... 37 7.8 Massively Parallel Sample Multiplexing .................................................................... 37

8 Sequence Generation and Potential Artifacts........................................................................... 38

8.1 Overview ..................................................................................................................... 38 8.2 Sequencing Methods and Instrumentation .................................................................. 39 8.3 Potential Artifacts and Troubleshooting ..................................................................... 41 8.4 Base Calling and Quality Scores ................................................................................. 45

9 Sequence Alignment, Assembly, and Evaluation .................................................................... 48

9.1 Sanger Sequencing ...................................................................................................... 50 9.2 Massively Parallel Sequencing ................................................................................... 54 9.3 Single Molecule Sequencing....................................................................................... 68

10 Validation and Verification ...................................................................................................... 68

10.1 General Considerations ............................................................................................... 68 10.2 Technology-Specific Considerations .......................................................................... 80

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Contents (Continued)

10.3 Application-Specific Considerations .......................................................................... 83 10.4 Revalidation ................................................................................................................ 89

11 Ongoing Quality Assurance and Quality Control .................................................................... 89

11.1 Definition and Purpose of Quality Assurance and Quality Control ............................ 89 11.2 Data Review ................................................................................................................ 90 11.3 Sequence Software Assessment and Review .............................................................. 94 11.4 Platform Quality Assurance ........................................................................................ 94 11.5 Assay Quality Control ................................................................................................ 94 11.6 Operator Competency ................................................................................................. 95 11.7 Confirmatory Testing .................................................................................................. 95 11.8 Proficiency Testing/External Quality Assessment and Alternate Methods for the

Independent Assessment of Laboratory Performance ................................................ 96

12 Interpreting and Reporting Results .......................................................................................... 97

12.1 Clinical Interpretation ................................................................................................. 97 12.2 Reports ...................................................................................................................... 102 12.3 Preservation of Records ............................................................................................ 104 12.4 Special Considerations .............................................................................................. 105

References ........................................................................................................................................... 106

Appendix. Example of Troubleshooting Guide for Sequencing Assays ............................................. 118

The Quality Management System Approach ...................................................................................... 120

Related CLSI Reference Materials ..................................................................................................... 122

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Foreword

Significant advances in clinical diagnostic sequencing prompted the development of the second edition of

this document. The original guideline focused primarily on the establishment and use of Sanger

sequencing in the clinical laboratory, which at the time was the principal means for the collection of DNA

sequence data. Since publication of the first edition, massively parallel sequencing (MPS) has become

part of the clinical laboratory repertoire. MPS is a catchall term that includes a number of technologies

that can generate a large amount of digital sequences. A feature that distinguishes MPS from Sanger

sequencing is the heavy reliance on informatics to process the raw data derived from the instrument into

interpretable DNA sequence. Laboratories have already begun to establish testing using MPS for heritable

conditions, cancer, and infectious diseases. Advances in other areas suggest that applications for analysis

of genome-wide methylation patterns, microbiomes, metagenomics, and transcriptome sequencing are

forthcoming.

This revised guideline provides additional details that address the implementation of MPS into the clinical

laboratory. In Section 5, users are initially oriented to the many sequencing technologies and currently

available applications; the contents of this section represent the technologies available at the time of this

publication, and users are encouraged to seek out recent reviews for the latest updates in this rapidly

evolving field. Information on the implementation of sequencing in the clinical laboratory has been

significantly expanded. Other sections have been added that discuss issues relevant to setup, running, and

QC of the instrumentation and considerations for informatics analysis. Test validation is discussed in

greater detail, and a separate section on QA and QC was also created. The final section that addresses the

reporting of results was revised primarily as a consequence of new guidance and resources that have

become available since publication of the previous edition of MM09. Sections that address Sanger

sequencing remain but have been updated as needed to reflect advances in practice. Sections addressing

specimen collection and preparation for analysis were consolidated into a much shorter section (see

Section 6) because these have become common laboratory practice and are covered in far greater detail in

other referenced CLSI documents. This revision is designed to provide guidance to experienced and

knowledgeable laboratory professionals to assist with the implementation of high-quality diagnostic

sequence analysis in the clinical laboratory.

Key Words

Capillary electrophoresis, clinical sequencing, dideoxy-terminators, electrophoresis, gel electrophoresis,

massively parallel sequencing, next generation sequencing, nucleic acid, polymerase chain reaction,

Sanger sequencing

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Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine;

Approved Guideline—Second Edition

1 Scope

The intended users of this guideline are clinical laboratories involved in the development, validation,

verification, and implementation of sequencing-based assays.

This guideline specifies recommendations for the sequencing process, including specimen collection and

handling, isolation of nucleic acid, amplification and sequencing of nucleic acids, and general

interpretation and reporting of genotyping results. It is the intent of this document to provide instruction

for verifying that the sequence obtained is accurate and suitable for subsequent interpretation; to address

general interpretation of the sequence; and to provide QA/QC considerations for each step of the process,

as appropriate. It is also intended to assist laboratories in generating appropriate and efficient validation

across sequencing methods and applications. Sanger-based DNA sequencing and general aspects of

massively parallel sequencing (MPS) are addressed in this guideline with specific examples.

This guideline:

Does not comprehensively address platform-specific issues, because sequencing technology is rapidly

evolving

Provides general guidance for interpreting sequencing results and does not address the medical

interpretation for a given patient, which is under the purview of the health care provider

Is relevant to germline, somatic, and microbiological applications in clinical settings

2 Introduction

Sequencing is an increasingly important tool for genotyping in molecular diagnostics. Sequencing is

routinely used in genotyping infectious disease organisms such as HIV and hepatitis C virus (HCV).

When typing tissue for transplantation, human leukocyte antigen (HLA) typing is also performed by

sequencing. There are also a variety of applications of sequencing for oncology and for diagnosing

heritable conditions. The widespread use of laboratory-developed, sequencing-based genotyping assays

and commercially available sequencing-based genotyping kits spurred the development of the original

guideline for the development, verification, validation, and implementation of sequencing-based assays.

The previous edition of this guideline focused on sequence analysis using dideoxy chain-terminating

chemistry and capillary electrophoresis (CE) instrumentation. In recent years, a number of new

technologies have been introduced commercially. One change that has occurred since the previous edition

of the guideline was published is that some instruments and assays have received regulatory approval or

clearance for Sanger or MPS clinical testing. For clinical tests that use these products, the process of test

validation and establishing QC parameters is simplified relative to a corresponding laboratory-developed

test. Additionally, there are an increasing number of clinical applications, especially for multigene panels

applied to genetic disease, for specific applications in which minor population variants can be clinically

important (eg, HIV tropism), and in oncology, where both broad coverage and low-level variant detection

can be of value. Emerging fields, such as epigenetics and the study of the microbiomes, suggest that

applications to additional complex clinical questions will only increase as sequencers are able to generate

more information at lower cost. This infusion of new technology requires a fresh look at some of the

subject matter covered for validation of CE sequencing applications, and also introduces new challenges

in the areas of platform validation and appropriate data analysis and management. This document was

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Clinical and Laboratory Standards Institute. All rights reserved. 2

revised during a time of flux, with several technologies currently undergoing rapid development in both

instrumentation and software.

3 Standard Precautions

Because it is often impossible to know what isolates or specimens might be infectious, all patient and

laboratory specimens are treated as infectious and handled according to “standard precautions.” Standard

precautions are guidelines that combine the major features of “universal precautions and body substance

isolation” practices. Standard precautions cover the transmission of all known infectious agents and thus

are more comprehensive than universal precautions, which are intended to apply only to transmission of

blood-borne pathogens. The Centers for Disease Control and Prevention address this topic in published

guidelines that focus on the daily operations of diagnostic medicine in human and animal medicine while

encouraging a culture of safety in the laboratory.1 For specific precautions for preventing the laboratory

transmission of all known infectious agents from laboratory instruments and materials and for

recommendations for the management of exposure to all known infectious diseases, refer to CLSI

document M29.2

4 Terminology

4.1 A Note on Terminology

CLSI, as a global leader in standardization, is firmly committed to achieving global harmonization

wherever possible. Harmonization is a process of recognizing, understanding, and explaining differences

while taking steps to achieve worldwide uniformity. CLSI recognizes that medical conventions in the

global metrological community have evolved differently in the United States, Europe, and elsewhere; that

these differences are reflected in CLSI, International Organization for Standardization (ISO), and

European Committee for Standardization (CEN) documents; and that legally required use of terms,

regional usage, and different consensus timelines are all important considerations in the harmonization

process. In light of this, CLSI’s consensus process for development and revision of standards and

guidelines focuses on harmonization of terms to facilitate the global application of standards and

guidelines.

In order to align the usage of terminology in this document with that of ISO and CLSI document QMS013

the terms preexamination, examination, and postexamination have replaced preanalytical, analytical, and

postanalytical, respectively, when referring to the testing phases within the path of workflow in a

laboratory.

4.2 Definitions

accuracy (of measurement) – closeness of agreement between a measured quantity value and a true

quantity value of a measurand (JCGM 200:2012)4; NOTE: For nucleic acid sequence analysis, the overall

accuracy refers to the closeness of the derived assembled sequence to the true sequence.

adapter (oligonucleotide adapter) – a short sequence of deoxynucleotides used to couple segments of

oligonucleotide.

alignment – the process of lining up two or more sequences for the purpose of assessing the percent

identity shared between sequences and/or constructing a contiguous sequence from overlapping smaller

segments.

all base accuracy – calculated by determining the percentage of the bases called that agree with the

expected base call in the reference sequence.

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The Quality Management System Approach Clinical and Laboratory Standards Institute (CLSI) subscribes to a quality management system approach in the

development of standards and guidelines, which facilitates project management; defines a document structure via a

template; and provides a process to identify needed documents. The quality management system approach applies a

core set of “quality system essentials” (QSEs), basic to any organization, to all operations in any health care

service’s path of workflow (ie, operational aspects that define how a particular product or service is provided). The

QSEs provide the framework for delivery of any type of product or service, serving as a manager’s guide. The QSEs

are as follows:

Organization Personnel Process Management Nonconforming Event Management

Customer Focus Purchasing and Inventory Documents and Records Assessments

Facilities and Safety Equipment Information Management Continual Improvement

MM09-A2 addresses the QSE indicated by an “X.” For a description of the other documents listed in the grid, please

refer to the Related CLSI Reference Materials section, beginning on page 122.

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Path of Workflow

A path of workflow is the description of the necessary processes to deliver the particular product or service that the

organization or entity provides. A laboratory path of workflow consists of the sequential processes: preexamination,

examination, and postexamination and their respective sequential subprocesses. All laboratories follow these

processes to deliver the laboratory’s services, namely quality laboratory information.

MM09-A2 addresses the clinical laboratory path of workflow steps indicated by an “X.” For a description of the

other documents listed in the grid, please refer to the Related CLSI Reference Materials section on the following

page.

Preexamination Examination Postexamination

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Related CLSI Reference Materials

EP07-A2 Interference Testing in Clinical Chemistry; Approved Guideline—Second Edition (2005). This document

provides background information, guidance, and experimental procedures for investigating, identifying, and

characterizing the effects of interfering substances on clinical chemistry test results.

EP15-A2 User Verification of Performance for Precision and Trueness; Approved Guideline—Second Edition

(2006). This document describes the demonstration of method precision and trueness for clinical laboratory

quantitative methods utilizing a protocol designed to be completed within five working days or less.

GP27-A2 Using Proficiency Testing to Improve the Clinical Laboratory; Approved Guideline—Second Edition

(2007). This guideline provides assistance to laboratories in using proficiency testing as a quality

improvement tool.

GP29-A2 Assessment of Laboratory Tests When Proficiency Testing Is Not Available; Approved Guideline—

Second Edition (2008). This document offers methods to assess test performance when proficiency testing

(PT) is not available; these methods include examples with statistical analyses. This document is intended for

use by laboratory managers and testing personnel in traditional clinical laboratories as well as in point-of-care

and bedside testing environments.

M29-A3 Protection of Laboratory Workers From Occupationally Acquired Infections; Approved Guideline—

Third Edition (2005). Based on US regulations, this document provides guidance on the risk of transmission

of infectious agents by aerosols, droplets, blood, and body substances in a laboratory setting; specific

precautions for preventing the laboratory transmission of microbial infection from laboratory instruments and

materials; and recommendations for the management of exposure to infectious agents.

MM01-A3 Molecular Methods for Clinical Genetics and Oncology Testing; Approved Guideline—Third Edition (2012). This document provides guidance for the use of molecular biological techniques for detection of

mutations associated with inherited medical disorders, somatic or acquired diseases with genetic associations,

and pharmacogenetic response.

MM03-A2 Molecular Diagnostic Methods for Infectious Diseases; Approved Guideline—Second Edition (2006). This guideline addresses topics relating to clinical applications, amplified and nonamplified nucleic acid

methods, selection and qualification of nucleic acid sequences, establishment and evaluation of test

performance characteristics, inhibitors, and interfering substances, controlling false-positive reactions,

reporting and interpretation of results, quality assurance, regulatory issues, and recommendations for

manufacturers and clinical laboratories.

MM05-A2 Nucleic Acid Amplification Assays for Molecular Hematopathology; Approved Guideline—Second

Edition (2012). This guideline addresses the performance and application of assays for gene rearrangement

and translocations by both polymerase chain reaction (PCR) and reverse-transcriptase PCR techniques, and

includes information on specimen collection, sample preparation, test reporting, test validation, and quality

assurance.

MM06-A2 Quantitative Molecular Methods for Infectious Diseases; Approved Guideline—Second Edition (2010). This document provides guidance for the development and use of quantitative molecular methods, such as

nucleic acid probes and nucleic acid amplification techniques of the target sequences specific to particular

microorganisms. It also presents recommendations for quality assurance, proficiency testing, and

interpretation of results.

MM13-A Collection, Transport, Preparation, and Storage of Specimens for Molecular Methods; Approved

Guideline (2005). This document provides guidance related to proper and safe biological specimen collection

and nucleic acid isolation and purification. These topics include methods of collection, recommended storage

and transport conditions, and available nucleic acid purification technologies for each specimen/nucleic acid

type. A CLSI-IFCC joint project.

CLSI documents are continually reviewed and revised through the CLSI consensus process; therefore, readers should refer to

the most current editions.

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Related CLSI Reference Materials (Continued)

MM14-A2 Design of Molecular Proficiency Testing/External Quality Assessment; Approved Guideline—Second

Edition (2013). This document provides guidelines for a quality proficiency testing/external quality

assessment program, including reliable databases; design control in the choice of materials and measurands;

good manufacturing processes; documentation procedures; complaint handling; corrective and preventive

action plans; and responsive timing of reports. A CLSI-IFCC joint project.

MM17-A Verification and Validation of Multiplex Nucleic Acid Assays; Approved Guideline (2008). This

guideline provides recommendations for analytic verification and validation of multiplex assays, as well as a

review of different types of biologic and synthetic reference materials.

MM18-A Interpretive Criteria for Identification of Bacteria and Fungi by DNA Target Sequencing; Approved

Guideline (2008). Sequencing DNA targets of cultured isolates provides a quantitative metric within which to

perceive microbial diversity, and can serve as the basis to identify microorganisms. This document is an effort

to catalyze the entry of molecular microbiology into clinical usage by establishing interpretive criteria for

microorganism identification.

`

MM19-A Establishing Molecular Testing in Clinical Laboratory Environments; Approved Guideline (2011). This

guideline provides comprehensive guidance for planning and implementation of molecular diagnostic testing,

including strategic planning, regulatory requirements, implementation, quality management, and special

considerations for the subspecialties of molecular genetics, infectious diseases, oncology, and

pharmacogenetics.

MM20-A Quality Management for Molecular Genetic Testing; Approved Guideline (2012). This document

provides guidance for implementing international quality management system standards in laboratories that

perform human molecular genetic testing for inherited or acquired conditions.

QMS01-A4 Quality Management System: A Model for Laboratory Services; Approved Guideline—Fourth Edition

(2011). This document provides a model for medical laboratories that will assist with implementation and

maintenance of an effective quality management system.

QMS02-A6 Quality Management System: Development and Management of Laboratory Documents; Approved

Guideline—Sixth Edition (2013). This document provides guidance on the processes needed for document

management, including creating, controlling, changing, and retiring a laboratory’s policy, process, procedure,

and form documents in both paper and electronic environments.

QMS06-A3 Quality Management System: Continual Improvement; Approved Guideline—Third Edition (2011). This guideline considers continual improvement as an ongoing, systematic effort that is an essential

component of a quality management system. A continual improvement program may consist of fundamental

processes and common supporting elements described in this guideline.

QMS11-A Management of Nonconforming Laboratory Events; Approved Guideline (2007). This guideline provides

an outline and the content for developing a program to manage a health care service’s nonconforming events

that is based on the principles of quality management and patient safety.

QMS12-A Development and Use of Quality Indicators for Process Improvement and Monitoring of Laboratory

Quality; Approved Guideline (2010). This document provides guidance on development of quality

indicators and their use in the medical laboratory.

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Power Forward with this Official Interactive Guide

Fundamentals for implementing a quality management system in the clinical laboratory.

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