November 2005

EP07-A2
Interference Testing in Clinical Chemistry;

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Approved Guideline—Second Edition

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This document provides background information, guidance,

and experimental procedures for investigating, identifying,
and characterizing the effects of interfering substances on
clinical chemistry test results.

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

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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

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and accept the resulting agreement.

Commenting on Documents

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

CLSI’s consensus process depends on experts who volunteer to serve as contributing authors and/or as
participants in the reviewing and commenting process. At the end of each comment period, the committee that
developed the document is obligated to review all comments, respond in writing to all substantive comments,
and revise the draft document as appropriate.

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

Appeals Process

If it is believed that an objection has not been adequately addressed, the process for appeals is documented in
the CLSI Administrative Procedures.
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 EP07-A2
Vol. 25 No. 27
ISBN 1-56238-584-4 Replaces EP7-A
ISSN 0273-3099 Vol. 22 No. 27
Interference Testing in Clinical Chemistry; Approved Guideline—
Second Edition
Volume 25 Number 27
Robert J. McEnroe, PhD
Mary F. Burritt, PhD

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Donald M. Powers, PhD
Douglas W. Rheinheimer, MT
Brian H. Wallace, PhD

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Abstract
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Clinical and Laboratory Standards Institute document EP07-A2—Interference Testing in Clinical Chemistry; Approved
Guideline—Second Edition is intended to promote uniformity in the evaluation of interference characteristics of clinical
laboratory measurement procedures. EP07 describes procedures for manufacturers to screen potentially interfering substances, to
quantify interference effects, and to confirm interference in patient samples. This document also describes procedures for clinical
laboratories to verify interference claims, and to investigate discrepant results caused by unsuspected interfering substances.
Detailed examples are given. EP07 also contains background information on interference testing concepts, tables of
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recommended test concentrations for analytes and potential interference, and data collection and analysis worksheets.

Clinical and Laboratory Standards Institute (CLSI). Interference Testing in Clinical Chemistry; Approved Guideline—Second
Edition. CLSI document EP07-A2 (ISBN 1-56238-584-4). Clinical and Laboratory Standards Institute, 950 West Valley Road,
Suite 2500, Wayne, Pennsylvania 19087 USA, 2005.

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 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:
[email protected]; Website: www.clsi.org.
Number 27 EP07-A2

Copyright ©2005 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
[email protected].

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 [email protected].

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Suggested Citation

CLSI. Interference Testing in Clinical Chemistry; Approved Guideline—Second Edition. CLSI document

August 1986

Approved Guideline
December 2002
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EP07-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2005.

Proposed Guideline

Approved Guideline—Second Edition

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November 2005
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ISBN 1-56238-584-4
ISSN 0273-3099

ii
Volume 25 EP07-A2

Contents

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

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

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

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

2 Introduction ................................................................................................................................ 1
2.1 Measurement Procedures .............................................................................................. 1
2.2 Concepts and Scientific Principles................................................................................ 2

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3 Standard Precautions .................................................................................................................. 4

4 Definitions ................................................................................................................................. 5

5 Decision Criteria for Interference Testing ................................................................................. 8

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5.1
5.2
5.3
5.4
5.5
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Clinical Acceptability Criteria ...................................................................................... 9
Statistical Significance and Power ................................................................................ 9
Analyte Test Concentrations ....................................................................................... 10
Potential Interfering Substances ................................................................................. 10
Interferent Test Concentrations................................................................................... 11
Quality Assurance and Safety .................................................................................................. 12
6.1 Training and Familiarization....................................................................................... 12
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6.2 Precision Verification ................................................................................................. 12
6.3 Trueness Verification .................................................................................................. 12
6.4 Carryover Assessment ................................................................................................ 12
6.5 Quality Control ........................................................................................................... 12
6.6 Safety and Waste Disposal.......................................................................................... 13
7 Estimation of Interference Characteristics ............................................................................... 13
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7.1 Interference Screen ..................................................................................................... 13

7.2 Characterization of Interference Effects ..................................................................... 20
7.3 Evaluating Combinations of Analyte and Interferent(s) ............................................. 26
8 Evaluating Interference Using Patient Specimens ................................................................... 26
8.1 Experimental Design................................................................................................... 26
8.2 Comparative Measurement Procedure ........................................................................ 27
8.3 Patient Populations ..................................................................................................... 27
8.4 Experimental Procedure .............................................................................................. 28
8.5 Data Analysis .............................................................................................................. 28
8.6 Interpretation of Results.............................................................................................. 31
9 Establishing, Validating, and Verifying Interference Claims .................................................. 31
9.1 Establishing Interference Claims ................................................................................ 32
9.2 Verifying Analytical Specificity ................................................................................. 34
9.3 Validating Analytical Specificity ................................................................................ 35
9.4 Verifying Interference and Specificity Claims ........................................................... 36

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Contents (Continued)
10 Investigating Discrepant Patient Results.................................................................................. 36
10.1 Verify System Performance ........................................................................................ 37
10.2 Evaluate Sample Quality ............................................................................................ 37
10.3 Confirm the Original Result........................................................................................ 37
10.4 Identify Potentially Interfering Substances ................................................................. 38
10.5 Determine the Probable Interferent ............................................................................. 38
10.6 Characterize the Interference ...................................................................................... 39
References ............................................................................................................................................. 40

Appendix A. Guidelines for Specific Measurement Procedures ......................................................... 43

Appendix B. Analyte Test Concentrations .......................................................................................... 46

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Appendix C. Interferent Test Concentrations ...................................................................................... 49

Appendix D. Interference Test Concentrations for Endogenous Analytes .......................................... 75

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Appendix E. Worksheets ..................................................................................................................... 79

Appendix F. Calculation of Replicates for Dose-Response Tests ....................................................... 87

Appendix G. Preparation of Test Solutions for Interference Testing .................................................. 90

Summary of Consensus Comments and Committee Responses ........................................................... 96

Summary of Consensus/Delegate Comments and Committee Responses .......................................... 101

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The Quality System Approach ............................................................................................................ 104

Related CLSI/NCCLS Publications .................................................................................................... 105

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Volume 25 EP07-A2

Interference Testing in Clinical Chemistry;

Approved Guideline—Second Edition

1 Scope
This document is intended to serve two purposes:

1) to assist manufacturers and other developers of laboratory measurement procedures in characterizing

the susceptibility of measurement procedures to interfering substances, by offering scientifically valid
experimental designs, by specifying the relevant substances and concentrations to be tested, and by
clarifying appropriate data analysis and interpretation, so that potential hazards can be evaluated and
meaningful interference claims may be provided to users; and

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2) to assist clinical laboratories in investigating discrepant results due to interfering substances, by
defining a systematic investigation strategy, by specifying data collection and analysis requirements,
and by promoting greater cooperation between laboratory users and manufacturers, so that new
interferences can be identified, disclosed, and ultimately eliminated.

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This guideline is intended for manufacturers of in vitro diagnostic medical devices and clinical
laboratories.

Manufacturers and other developers of laboratory measurement procedures are responsible for
characterizing the analytical performance of their procedures and analyzing hazards to patients caused by
errors due to interfering substances. Manufacturers are required to provide information about interference
susceptibility to those who use their systems. NOTE: The term “manufacturer,” for the purpose of this
document, is used to mean anyone that develops a measurement procedure for use in a clinical laboratory.
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Clinical laboratories are responsible for ensuring that measurement procedures are specific enough to
meet the needs of their physician clients. Laboratories should also investigate discrepant results, identify
interfering substances, and provide objective feedback to the manufacturers who supply their analysis
systems.
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2 Introduction

2.1 Measurement Procedures

Any measurement procedure, quantitative or qualitative, may be subject to interference. This document is
written for a broad spectrum of measurement procedures and analyzers. Modification may be necessary to
accommodate the particular characteristics of the procedure being evaluated. Two specific method
principles (i.e., separation techniques and immunochemical measurement procedures) are discussed in
Appendix A.

2.1.1 Specimen Type

Interferences with measurement procedures that use serum, plasma, whole blood, cerebrospinal fluid,
urine, and most other body fluids may be evaluated using this guideline.

2.1.2 Interfering Substances

Potentially interfering substances may originate from the following endogenous and exogenous sources:

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Clinical and Laboratory Standards Institute. All rights reserved. 1
Number 27 EP07-A2

 metabolites produced in pathological conditions, such as diabetes mellitus, multiple myeloma,

cholestatic hepatitis, etc.;

 compounds introduced during patient treatment, such as drugs, parenteral nutrition, plasma
expanders, anticoagulants, etc.;

 substances ingested by the patient, such as alcohol, drugs of abuse, nutritional supplements, various
foods and drink, etc.;

 substances added during sample preparation, such as anticoagulants, preservatives, stabilizers, etc.;

 contaminants inadvertently introduced during sample handling from sources such as hand cream,
powdered gloves, serum separators, collection tube stoppers, etc.; and

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the sample matrix itself, such as chemical and physical properties that differ from the ideal fresh
sample.13-16

2.2 Concepts and Scientific Principles

2.2.1

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Contribution of Interference to Inaccuracy

Inaccuracy (total analytical error) consists of three principal contributors: imprecision, method-specific
bias, and sample-specific bias.17,18 Measurement procedure evaluations frequently estimate only the first
two. Sample-specific bias (i.e., interference) is often viewed as an isolated problem with specific samples,
rather than as a quantifiable characteristic of the procedure. From the standpoint of an evaluation,
susceptibility to interference causes both systematic and random error, both of which can be quantified
statistically as components of inaccuracy (total analytical error).19,20
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 For a given patient population, the average concentration of interfering substances in the samples
may cause a systematic bias, which will be included in the estimate of bias. Individual deviations
from this average bias contribute to the total random error observed in a comparison to a more
specific measurement procedure. For some procedures, random interference effects exceed
imprecision as the dominant source of random error.
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 For an individual patient, interfering substances cause a bias dependent on their concentrations in the
patient’s specimen. The bias changes as the interferent concentration changes (e.g., due to clearance
or metabolism). The resulting change in bias could be erroneously interpreted as a change in patient
condition.

2.2.2 Clinical Relevance

In laboratory medicine, interference has to be viewed from a clinical perspective. Clinical relevance
determines whether an analytical effect is considered interference. The form of the analyte intended to be
measured and its concentration basis must be clearly defined.

Paradoxically, analytical results from some measurement procedures may reflect the true analyte
concentrations, but not necessarily the clinically relevant values. For example, flame photometry and
indirect potentiometry correctly measure the total concentration of sodium in an aliquot of plasma,
regardless of the lipid concentration. However, if the lipid concentration is high, these procedures will
falsely indicate hyponatremia in a patient with proper electrolyte balance. Direct potentiometry correctly
reports normal sodium in this case, because it responds to sodium activity in the plasma water fraction,
which is what the body regulates. Thus, overestimating the total sodium in the sample is appropriate from

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2 Clinical and Laboratory Standards Institute. All rights reserved.
Number 27 EP07-A2

The Quality 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 approach is based on the model presented in the
most current edition of CLSI/NCCLS document HS1—A Quality Management System Model for Health Care. The
quality management system approach applies a core set of “quality system essentials” (QSEs), basic to any
organization, to all operations in any healthcare service’s path of workflow (i.e., 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 quality system essentials (QSEs) are:

Documents & Records Equipment Information Management Process Improvement

Organization Purchasing & Inventory Occurrence Management Service & Satisfaction
Personnel Process Control Assessment Facilities & Safety

EP07-A2 addresses the quality system essentials (QSEs) indicated by an “X.” For a description of the other

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documents listed in the grid, please refer to the Related CLSI/NCCLS Publications section on the following page.
Purchasing &

Improvement
Organization

Management

Management
Information

Assessment

Satisfaction

Facilities &
Occurrence
Documents

Equipment
& Records

Service &
Personnel

Inventory

Control
Process

Process
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Safety
X
C3
C24
EP5
EP9
EP14
Adapted from CLSI/NCCLS document HS1—A Quality Management System Model for Health Care.
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104 Clinical and Laboratory Standards Institute. All rights reserved.
Volume 25 EP07-A2

Related CLSI/NCCLS Publications*

C3-P4 Preparation and Testing of Reagent Water in the Clinical Laboratory; Proposed Guideline—Fourth
Edition (2005). This document provides guidance on water purified for clinical laboratory use; methods for
monitoring water quality and testing for specific contaminants; and water system design considerations.

C24-A2 Statistical Quality Control for Quantitative Measurements: Principles and Definitions; Approved
Guideline—Second Edition (1999). This guideline provides definitions of analytical intervals, planning of
quality control procedures, and guidance for quality control applications.

EP5-A2 Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline—

Second Edition (2004). This document provides guidance for designing an experiment to evaluate the
precision performance of quantitative measurement methods; recommendations on comparing the resulting
precision estimates with manufacturers’ precision performance claims and determining when such
comparisons are valid; as well as manufacturers’ guidelines for establishing claims.

EP9-A2 Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline—Second Edition
(2002). This document addresses procedures for determining the bias between two clinical methods, and the

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design of a method comparison experiment using split patient samples and data analysis.

EP14-A2 Evaluation of Matrix Effects; Approved Guideline—Second Edition (2005). This document provides
guidance for evaluating the bias in analyte measurements that is due to the sample matrix (physiological or
artificial) when two measurement procedures are compared.

HS1-A2

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A Quality Management System Model for Health Care; Approved Guideline—Second Edition (2004).
This document provides a model for providers of healthcare services that will assist with implementation and
maintenance of effective quality management systems.
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*
Proposed-level documents are being advanced through the Clinical and Laboratory Standards Institute consensus process;
therefore, readers should refer to the most recent editions.
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Clinical and Laboratory Standards Institute. All rights reserved. 105
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