H59-A Quantitative Ddimer For The Exclusion of VTE

H59-A
Vol. 31 No. 6
Replaces H59-P
Vol. 30 No. 9
Quantitative D-dimer for the Exclusion of

Venous Thromboembolic Disease;
Approved Guideline
This document provides guidelines regarding the use of D-dimer in exclusion of venous
thromboembolism (VTE) including a description of the value of clinical determination of
the pretest probability of VTE; the proper collection and handling of the specimen; assays
used for D-dimer analysis; determination of the threshold for exclusion of VTE;
interpretation of test results; and aspects of regulatory and accreditation requirements.
A guideline for global application developed through the Clinical and Laboratory
Standards Institute consensus process.
Licensed to: Paul D\'Orazio, PhD Instrumentation Laboratory

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H59-A
ISBN 1-56238-747-2
Volume 31 Number 6 ISSN 0273-3099
Quantitative D-dimer for the Exclusion of Venous Thromboembolic
Disease; Approved Guideline
John D. Olson, MD, PhD C. Alex McMahan, PhD

Dorothy M. Adcock, MD Thomas J. Prihoda, PhD
Theresa Ambrose Bush, PhD, RAC, DABCC Alicia Rico-Lazarowski, H(ASCP)cm
Philippe de Moerloose, MD Miquel Sales
Chris Gardiner, FIBMS, MSc, PhD Linda Stang, MLT
Valerie R. Ginyard, BSMT(ASCP) Kathleen Trumbull, MS, MT(ASCP)
Marc Grimaux, PhD Elizabeth M. Van Cott, MD
Thomas Wissel, PhD
Abstract
D-dimer is a product of fibrinolysis that is assayed in the blood. It is elevated following intravascular thrombosis, disseminated
intravascular coagulation, and other conditions that can cause fibrin generation. Assay of D-dimer is a useful tool when
evaluating patients with possible venous thromboembolism (VTE), as the absence of D-dimer is helpful in excluding VTE.
Clinical and Laboratory Standards Institute document H59-A—Quantitative D-dimer for the Exclusion of Venous
Thromboembolic Disease; Approved Guideline provides guidance regarding the use of D-dimer in exclusion of VTE including a
description of the value of clinical determination of the pretest probability of VTE; the proper collection and handling of the
specimen; assays used for D-dimer analysis; determination of the threshold for exclusion of VTE; interpretation of test results;
and aspects of regulatory and accreditation requirements. The guideline is provided for use by laboratorians, manufacturers of D-
dimer assays, clinicians who use the D-dimer for VTE exclusion, and accrediting and regulatory agencies.
Clinical and Laboratory Standards Institute (CLSI). Quantitative D-dimer for the Exclusion of Venous Thromboembolic Disease;
Approved Guideline. CLSI document H59-A (ISBN 1-56238-747-2). Clinical and Laboratory Standards Institute, 940 West
Valley Road, Suite 1400, Wayne, Pennsylvania 19087 USA, 2011.
The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through
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customerservice@clsi.org; Website: www.clsi.org

Number 6 H59-A
Copyright ©2011 Clinical and Laboratory Standards Institute. Except as stated below, neither this
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Suggested Citation
CLSI. Quantitative D-dimer for the Exclusion of Venous Thromboembolic Disease; Approved Guideline.
CLSI document H59-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2011.
Proposed Guideline
April 2010
Approved Guideline
March 2011
ISBN 1-56238-747-2
ISSN 0273-3099
ii D\'Orazio, PhD Instrumentation Laboratory

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Volume 31 H59-A
Committee Membership
Consensus Committee on Hematology

Dorothy M. Adcock, MD Josephine M. Bautista, MS, Ian Giles, MD
Chairholder MT(ASCP) Sysmex America, Inc.
Esoterix Coagulation FDA Center for Devices and Mundelein, Illinois, USA
Englewood, Colorado, USA Radiological Health
Rockville, Maryland, USA Hans Hoffmann, PhD
Stephen Kitchen, FIBMS, PhD Abbott GmbH
Vice-Chairholder Douglas J. Christie, PhD, FAHA Wiesbaden, Germany
Royal Hallamshire Hospital Siemens Healthcare Diagnostics
Sheffield, United Kingdom Newark, Delaware Powers Peterson, MD
Weill Cornell Medical College
Maria J. Arroz, MD Emmanuel Favaloro, PhD in Qatar
Hospital S. Francisco Xavier ICPMR, Westmead Hospital Education City, Doha, Qatar
Lisbon, Portugal New South Wales, Australia
Document Development Committee on Quantitative D-dimer
John D. Olson, MD, PhD Kathleen Trumbull, MS, Lois M. Schmidt, DA

Chairholder MT(ASCP) Vice President, Standards
University of Texas Health Center Instrumentation Laboratory Development
San Antonio, Texas, USA Bedford, Massachusetts, USA
Jennifer K. Adams, MT(ASCP),
Dorothy M. Adcock, MD Elizabeth M. Van Cott, MD MSHA
Esoterix Coagulation Massachusetts General Hospital Staff Liaison
A LabCorp Company Boston, Massachusetts, USA
Englewood, Colorado, USA David E. Sterry, MT(ASCP)
Thomas Wissel, PhD Project Manager
Valerie R. Ginyard, BSMT(ASCP) Siemens Healthcare Diagnostics
FDA Center for Devices and Marburg, Germany Melissa A. Lewis, ELS
Radiological Health Editorial Manager
Rockville, Maryland, USA Staff
Megan P. Larrisey, MA
Marc Grimaux, PhD Clinical and Laboratory Standards Assistant Editor
Diagnostica Stago Institute
Gennevilliers, France Wayne, Pennsylvania, USA
Acknowledgment
CLSI and the Consensus Committee on Hematology gratefully acknowledge the following individuals for
their contributions during the development of this approved-level document:
Theresa Ambrose Bush, PhD, RAC, DABCC Thomas J. Prihoda, PhD
Roche Diagnostics University of Texas Health Science Center
Indianapolis, Indiana, USA San Antonio, Texas, USA
Philippe de Moerloose, MD Alicia Rico-Lazarowski, H(ASCP)CM

University Hospital of Geneva bioMérieux
Geneva, Switzerland Durham, North Carolina, USA
Chris Gardiner, FIBMS, MSc, PhD Miguel Sales

University College London Hospitals Biokit SA
London, United Kingdom Barcelona, Spain
C. Alex McMahan, PhD Linda Stang, MLT

University of Texas Health Science Center University of Alberta Hospital
San Antonio, Texas, USA Edmonton, Alberta, Canada
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Volume 31 H59-A
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 ....................................................................................... 7
5 Overview of D-dimer Testing .................................................................................................... 7
5.1 Venous Thromboembolism Background ...................................................................... 7
5.2 D-dimer Measurement .................................................................................................. 8
5.3 Problems With D-dimer Measurement ......................................................................... 9
5.4 Harmonization vs Standardization ................................................................................ 9
6 Patient Evaluation and Pretest Probability ............................................................................... 10
6.1 Limitations Related to Patient Conditions or Therapies ............................................. 12
7 Specimen Collection and Processing ....................................................................................... 13
7.1 Patient Variables/Preparation Before Collection ........................................................ 13
7.2 Specimen Collection ................................................................................................... 14
7.3 Specimen Processing, Transport, and Storage ............................................................ 14
7.4 Interferences................................................................................................................ 15
8 Assay Methodologies ............................................................................................................... 15
8.1 Quantitative Sandwich Assays.................................................................................... 15
8.2 Quantitative Microparticle Agglutination ................................................................... 16
8.3 Semiquantitative Microparticle Agglutination Methods............................................. 16
8.4 Point-of-Care Testing ................................................................................................. 16
8.5 Other Technologies ..................................................................................................... 17
9 Assay Characteristics, Reference Interval, and Threshold for Exclusion of Venous
Thromboembolism ................................................................................................................... 17
9.1 Recommended Assay Characteristics ......................................................................... 17
9.2 Reference Interval ....................................................................................................... 18
9.3 Establishing the Threshold for Venous Thromboembolism Exclusion:
Comparison to Clinical and Imaging Studies ............................................................. 18
9.4 Methods That a Laboratory May Use to Determine the Threshold for
Exclusion of Venous Thromboembolism ................................................................... 19
9.5 Satisfying Regulatory Requirements .......................................................................... 20
10 Interpretation of Results ........................................................................................................... 20
10.1 Result Reporting ......................................................................................................... 21
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Contents (Continued)
References ............................................................................................................................................. 23
Appendix. US Food and Drug Administration Clinical Study Design: An Example ........................... 28
The Quality Management System Approach ........................................................................................ 30
Related CLSI Reference Materials ....................................................................................................... 31
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Volume 31 H59-A
Foreword
Since the 1960s, clinicians have measured the products of plasmin action on fibrin, in the form of
fibrin(ogen) degradation products (FDPs), as an indicator of intravascular fibrinolysis. Initial use of the
FDP assay was to assist in the evaluation and monitoring of patients with disseminated intravascular
coagulation (DIC). In the mid-1980s, the first monoclonal antibody-based assays for D-dimer, a specific
FDP, were described, providing an assay with greater specificity for fibrin.1 Fibrin is the basic structural
molecule constituting the thrombus, and D-dimer is the smallest crosslinked degradation product of
crosslinked fibrin. Because the D-dimer test is very sensitive, with the concentration being elevated
whenever fibrin, formed in the vasculature, is being degraded, the test has generally replaced the assay for
FDP in the clinical setting.
Many clinical conditions are associated with increased blood concentrations of D-dimer. Some of these
include venous thromboembolism (VTE), arterial thrombosis (including myocardial infarction and
stroke), DIC, association with recurrent thrombotic risk following anticoagulation, the postoperative state,
significant liver disease, malignancy, and normal pregnancy.2 However, the current clinical use of the D-
dimer assay is primarily for the diagnosis and monitoring of DIC and for the exclusion of VTE, in
particular, deep vein/venous thrombosis, and pulmonary embolism/embolus, which are the focuses of this
document.
Patients who present with signs and symptoms that may be caused by VTE require evaluation to exclude
or confirm the diagnosis of a thrombus. Objective confirmation is needed because venous thrombosis is
not reliably diagnosed on clinical grounds alone; omission of therapy if a thrombus is missed could be
life-threatening, and the administration of anticoagulant therapy also carries risk. The most reliable of
such tests are imaging studies that are time-consuming and expensive to perform. Knowing that the
presence of an acute intravascular thrombus is associated with fibrinolysis and elevation of D-dimer in the
blood has led to the concept that below a certain threshold D-dimer may be an effective way to exclude
VTE and proceed without performing the imaging studies. If effective, such an approach may, of course,
provide savings in time and resources. However, there are many possible causes of elevations of the D-
dimer and using the test for VTE exclusion in these settings may actually lead to imaging studies of
limited value. The approach also carries the inherent risk of incorrectly excluding VTE, placing the
patient at risk of thrombus growth or embolus without appropriate anticoagulation, which is a potentially
life-threatening situation. Thus, the power of the D-dimer test for exclusion of VTE must be very high to
provide the best protection for the patient and it is best applied only in settings where known alternative
causes of elevations are not present.3
The development of commercial assays for D-dimer has grown rapidly, approaching 24 at the time of this
writing. Considerable variability has been reported among these commercial offerings. One major source
of variability is in the units reported. Quantitative D-dimer results are provided in mass units. As these
assays have evolved, two different types of units of significantly different molecular weight have been
used to represent D-dimer, the fibrinogen equivalent unit at 340 kDa and the D-dimer unit at 195 kDa.
Adding to the complexity of reporting these values is variability in the magnitude of the units reported,
eg, nanograms per milliliter, micrograms per milliliter, and micrograms per liter. This variability in both
the type and magnitude of units contributes to the general inconsistency among assays performed with
different methods in different laboratories, as do the differences in the specificities of the antibodies used.
This inconsistency has led to confusion in some laboratories, especially when the threshold for the
exclusion of VTE must be set. This issue of the type of units is not well recognized by laboratorians or
clinicians and is often ignored in some publications by recognized experts in the field.
In addition to the technical issues in the analytical (examination) aspects of the test, when applied to the
exclusion of VTE, the clinical evaluation of the pretest probability of VTE, specimen collection and
handling, and the reporting and interpretation of results are also critical elements of the use of the test in
this clinical setting.
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Number 6 H59-A
CLSI document H59-A reviews the clinical application of the D-dimer assay for the exclusion of VTE for
nonhospitalized, ambulatory patients. Use of D-dimer for the exclusion of VTE on hospitalized patients is
less effective, as most of these patients have an elevated D-dimer concentration because of
immobilization, surgery, or other conditions. The purpose of this document is to focus on the
preanalytical, analytical, and postanalytical (preexamination, examination, and postexamination) elements
of the use of the D-dimer test as it is applied to the exclusion of VTE. It addresses the evaluation of the
patient in the determination of the probability of VTE; specimen collection, transport, and processing;
analytical (examination) methods (measurement procedures/analytical method); reference intervals;
establishment and reporting of the threshold for exclusion of VTE; and interpretation of results. It is
intended to provide valuable guidance to laboratorians, clinicians, manufacturers, and regulators as the
use of the D-dimer assay for the exclusion of VTE continues to evolve.
Key Words
D-dimer, deep vein/venous thrombosis (DVT), negative predictive value (NPV), pretest probability
(PTP), pulmonary embolism/embolus (PE), quantitative D-dimer, threshold, thrombosis, venous
thromboembolism (VTE)
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Volume 31 H59-A
Quantitative D-dimer for the Exclusion of Venous Thromboembolic Disease;

Approved Guideline
1 Scope
This document provides guidelines regarding preanalytical, analytical, and postanalytical
(preexamination, examination, and postexamination) elements of testing including, but not limited to:
• A description of the value of clinical determination of the pretest probability (PTP) of venous
thromboembolism (VTE)
• The proper collection and handling of the specimen
• Assays used for D-dimer analysis
• Establishment of the threshold for exclusion of VTE and its interpretation related to the reference
interval (RI)
• Interpretation of test results
• Aspects of regulatory and accreditation requirements
The guideline is intended for clinical laboratorians and laboratory directors, for manufacturers of the
methods used to perform the test, for clinicians with an interest in the laboratory elements of the tests,
and for regulatory and accrediting agencies overseeing the use of D-dimer for this purpose.
This guideline is not intended for use by patients with clinical conditions that require D-dimer evaluation.
Patients reading this document are encouraged to discuss its content with their health care providers.
Issues of intermethod standardization or the development of calibrators for standardization are discussed;
however, guidelines regarding standardization and calibration are beyond the scope of this document. The
document does not address other clinical settings in which the measurement of D-dimer may be clinically
useful, including diagnosis and monitoring of overt and nonovert disseminated intravascular coagulation
(DIC); risk of recurrence of VTE following the completion of anticoagulant therapy; detection of occult
malignancy; staging or risk stratification of diagnosed malignancy; risk of future myocardial infarction in
patients presenting with chest pain; and evaluation for subarachnoid hemorrhage.2 Studies have
demonstrated some value in the combined use of the D-dimer and ultrasonography in the exclusion of
VTE. However, the focus of this document is the value of D-dimer to potentially avoid the need for
imaging studies. The combined use of D-dimer with imaging studies in the evaluation of VTE is not
addressed.
2 Introduction
Because of the potential to efficiently evaluate patients with VTE, many assays have been developed to
measure D-dimer in the blood. The assays vary in their sensitivity to D-dimer, the type and magnitude of
units used to report results, the type of specimen used, and other characteristics. Experience has
uncovered a number of limitations of the test as it applies to the exclusion of VTE.
The purpose of this document, based on currently available evidence, is to provide guidelines regarding
the use of the D-dimer assay for the exclusion of VTE. Elements of the clinical evaluation of the patient,
specimen collection and processing, analytical (examination) methods, developments of thresholds for the
exclusion of VTE, and the interpretation of results are addressed. It is important to remember that the data
regarding the use of clinical PTP and the D-dimer test for exclusion were developed in the clinical setting
of deep vein/venous thrombosis (DVT) and pulmonary embolism/embolus (PE). Application of these
guidelines in evaluation of other thrombotic events is not recommended and may be misleading. Patients
with distal DVT will have a normal D-dimer 35% of the time and the test cannot be used to avoid
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PhD Instrumentation All rights reserved.
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ultrasound evaluation.4 All patients with suspected distal DVT require ultrasound evaluation. D-dimer
testing used in concert with lower extremity ultrasound evaluation may be helpful.
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. Standard and universal precaution guidelines are available from the US Centers
for Disease Control and Prevention.5 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 M29.6
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.
To align the use of terminology in this document with that of ISO, the term precision is defined as the
“closeness of agreement between indications or measured quantity values obtained by replicate
measurements on the same or similar objects under specified conditions.” As such, it cannot have a
numerical value but may be determined qualitatively as high, medium, or low. For its numerical
expression, the term imprecision is used, which is the “dispersion of independent results of measurements
obtained under specified conditions.” In addition, one other component of precision is defined in H59-A,
reproducibility, which is “measurement precision (closeness of agreement between indications or
measured quantity values obtained by replicate measurements on the same or similar objects under
specified conditions) under reproducibility conditions of measurement (condition of measurement, out of
a set of conditions that includes different locations, operators, measuring systems, and replicate
measurements on the same or similar objects).”
The term measurand (quantity intended to be measured) is used in combination with the term analyte
(component represented in the name of a measurable quantity) when its use relates to a biological
fluid/matrix; and the term measurement procedure is combined with analytical method for a set of
operations, used in the performance of particular measurements according to a given method.
4.2 Definitions
aid in diagnosis – as defined by the US Food and Drug Administration, an adjunct assay that is used in
conjunction with clinical indications. The assay’s threshold value has been validated and device
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Volume 31 H59-A
performance, such as negative predictive value, has been demonstrated. The assay’s relative sensitivity
and specificity may or may not be established.
analyte – component represented in the name of a measurable quantity (ISO 17511)7; NOTE 1: In the
type of quantity “mass of protein in 24-hour urine,” “protein” is the analyte. In “amount of substance of
glucose in plasma,” “glucose” is the analyte. In both cases, the long phrase represents the measurand
(ISO 17511)7; NOTE 2: In the type of quantity “catalytic concentration of lactate dehydrogenase
isoenzyme 1 in plasma,” “lactate dehydrogenase isoenzyme 1” is the analyte (ISO 18153).8
anticoagulant – an agent, natural or pharmacological, that inhibits clotting of blood or plasma.
calibration – operation that, under specified conditions, in a first step, establishes a relation between the
quantity values with measurement uncertainties provided by measurement standards and corresponding
indications with associated measurement uncertainties and, in a second step, uses this information to
establish a relation for obtaining a measurement result from an indication (ISO/IEC Guide 99)9; NOTE:
In the context of this document, the process of testing and adjustment of an instrument, kit, or test system,
to provide a known relationship between the measurement response and the value of the substance being
measured by the test procedure (42 CFR 493.1217).10
CE marking – symbolizes the conformity of the product with the applicable Community requirements
imposed on the manufacturer. The CE marking affixed to products is a declaration by the person
responsible that the product conforms to all applicable Community provisions, and the appropriate
conformity assessment procedures have been completed.11
chemiluminescence immunoassay – nonenzymatic chemical reaction that emits light, the amount of
which can be related to the concentration of the analyte being measured.
deep vein/venous thrombosis (DVT) – an intravenous thrombus in a deep vein, usually in the proximal
legs or pelvis, but may also occur in an upper extremity.
distal deep vein/venous thrombosis – refers to intravenous thrombosis in a lower extremity affecting the
veins distal to the popliteal fossa.
enzyme-linked immunosorbent assay (ELISA) – a ligand binding assay in which a binding molecule
(often an antibody or antigen) is attached to a solid-phase surface, commonly a microtiter plate well. The
antigen containing fluid is added to the antibody/antigen reaction. The extent of binding is determined by
the activity of an enzyme that is conjugated with secondary antibodies that are added to the reaction along
with the appropriate substrate. Color or light is used to measure the amount of product.
exclusion of venous thromboembolism – a claim that can apply to any method, the results of which can
reliably exclude venous thromboembolism (VTE); NOTE 1: Regarding a D-dimer reagent, studies must
demonstrate that the negative predictive value (NPV), sensitivity, and coefficient of variation at the
threshold have sufficient power to exclude VTE when the test is applied to patients judged to have a low
or intermediate probability of VTE determined using a pretest probability (PTP) scoring algorithm;
NOTE 2: As defined by the US Food and Drug Administration, a D-dimer assay with an exclusionary
claim is used in conjunction with a PTP assessment to exclude the presence of a pulmonary
embolism/embolus and/or a deep vein/venous thrombosis. In addition to validating the assay’s threshold
and establishing assay sensitivity, specificity, and NPVs compared with imaging studies, the clinical
study must include patient follow-up to obtain clearance.
fluorescence energy transfer immunoassay – a type of sandwich fluorescence immunoassay of an

antigen with multivalent antigenic determinants in which antibodies are labeled with donor and acceptor
fluorescence markers; NOTE 1: The reaction of at least two antibodies with the targeted antigen,
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Number 6 H59-A
resulting in the approximation of these two determinants closer than a critical distance of 50 to 70
Angstroms that quenches the fluorescence; NOTE 2: The reduction in fluorescence emission is
proportional to the antigen concentration.
fluorescence immunoassay – a type of immunoassay in which the antigen or, as in the case of D-dimer,
antibody is labeled with a fluorescent compound that emits light at a wavelength when excited by a light
at a different wavelength or by a chemical reaction.
fluorescence resonance energy transfer – a type of fluorescent immunoassay, in which a donor

molecule (which is usually the substrate) is excited, electronically or by an external light source, and
instead of emitting light, the excitation energy transfers to a nearby acceptor molecule (which is the
product); NOTE: The excited states of one or both of the donor and acceptor can decay with fluorescence
emission. When energy transfer is observed, a reduction in the emission intensity of the donor/substrate
with a concomitant increase in the emission intensity from the acceptor/product is observed. The intensity
of the longer wavelength emission from acceptor/product is proportional to its amount.
heterogeneous immunoassay – an immunoassay that requires one or more steps to separate the bound
from the free indicator.
heterophile antibody – antibody produced against poorly defined antigens that react with
immunoglobulins from two or more species, or exhibit rheumatoid factor activity and bind to the Fc
portion of human or animal immunoglobulins.
homogeneous immunoassay – an immunoassay that does not require a step for the separation of the
bound and free indicator.
immunoassay (immune assay) – an analytical procedure in which antibodies are used to detect or
quantify the corresponding antigen; NOTE: Immunoassays can be used to quantify D-dimer and some
methods that may be employed are nonlabeled immunoassays/microparticle agglutination immunoassays,
indicator-labeled immunoassays, and sandwich immunoassays.
imprecision – dispersion of independent results of measurements obtained under specified conditions;

NOTE: It is expressed numerically as standard deviation or coefficient of variation.
incidence – an expression of the rate at which a certain event occurs, for example, the number of new
cases of a specific disease occurring during a specific period; NOTE 1: The number of instances of
illness or other outcomes commencing, or of persons falling ill, during a given period in a specified
population; NOTE 2: More generally, the number of new events, eg, new cases of a disease in a defined
population, within a specified period of time.
indicator-labeled immunoassays – a group of immunoassays in which the antibodies are labeled with an
indicator (commonly enzyme, fluorescent, chemiluminescent, radioisotope). The positive reaction
between labeled antibodies and/or antigens is detected by a system that measures some property of the
indicator molecule. These are mostly heterogeneous assays that require the separation of the bound from
the free fractions; NOTE: Examples include enzyme-linked immunosorbent assays, chemiluminescence
immunoassays, fluorescence immunoassays, and radioimmunoassays.
luminescence oxygen channeling immunoassay – a type of chemiluminescent assay that uses two types
of ligand-coated latex beads. One of the beads is a chemiluminescent bead in which an olefin is dissolved;
NOTE: The olefin can react rapidly with singlet oxygen. The reaction yields a dioxetane that
spontaneously decomposes with efficient emission of light. This can be used as a homogeneous assay.
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manufacturer – natural or legal person with responsibility for the design, manufacture, packaging, or
labeling of a medical device, assembling a system, or adapting a medical device before it is placed on the
market or put into service, regardless of whether these operations are carried out by that person or on that
person’s behalf by a third party (ISO 14971)12; NOTE: Provisions of national or regional regulations may
apply to the definition of manufacturer.
measurand – quantity intended to be measured (ISO/IEC Guide 99).9
measurement procedure – detailed description of a measurement according to one or more measurement

principles and to a given measurement method, based on a measurement model and including any
calculation to obtain a measurement result (ISO/IEC Guide 99)9; NOTE: A measurement procedure is usually
documented in sufficient detail to enable an operator to perform a measurement (ISO/IEC Guide 99).9
negative predictive value (NPV) – the likelihood that an individual with a negative test result does not
have the disease, or other characteristic, that the test is designed to detect; NOTE 1: This varies with the
prevalence of the disease in the population tested; NOTE 2: From predictive value theory, the proportion
of all negative tests that are true negatives.
nonlabeled immunoassays//microparticle agglutination immunoassay – a type of assay that is based

on agglutination, detecting soluble antigen. In this assay, microparticles (latex, polystyrene) coated with
antibodies agglutinate in the presence of antigen and can be detected by turbidimetric or nephelometric
techniques. This is a homogeneous assay that does not require the separation of the bound and free
fractions.
positive predictive value (PPV) – the likelihood that an individual with a positive test result has a
particular disease, or characteristic, that the test is designed to detect; NOTE 1: This varies with the
prevalence of the disease in the population tested; NOTE 2: From predictive value theory, the proportion
of positive tests that are true positives.
precision (measurement) – closeness of agreement between indications or measured quantity values

obtained by replicate measurements on the same or similar objects under specified conditions (ISO/IEC
Guide 99). 9
pretest probability (PTP) – the a priori probability of a particular clinical state before results of a
diagnostic test are known; NOTE 1: This probability may be obtained by estimates of the prevalence of a
disease in a population or by a clinical estimate of the probability of a disease existing in a given patient;
NOTE 2: An evaluation of clinical features (history, signs, and symptoms) that collectively assist in
predicting the outcome of a test or procedure. For D-dimer, concentrations are described as low,
intermediate or moderate (unlikely), and high (likely). Intermediate and moderate are used
interchangeably and in this document, the term intermediate is used for consistency.
prevalence – the frequency of a condition of interest expressed as a percentage (number fraction

multiplied by 100) of the total number of individuals (those with the condition plus those without the
condition of interest) in the population under study (see CLSI document EP12).13
proximal deep vein/venous thrombosis – intravenous thrombus occurring in the iliac, femoral, or
popliteal veins. These veins are in and proximal to the popliteal fossa but do not include superficial
(subcutaneous) veins.
pulmonary embolism/embolus (PE) – a material that travels in the venous bloodstream until it lodges in
the constriction of the pulmonary arterial system. In this document the issue is thromboembolism.
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qualitative – characterization applied to laboratory tests that detect and/or identify a particular analyte,
constituent, or condition; NOTE: This term is applied to tests that detect whether a particular analyte,
constituent, or condition is present or absent, and is sometimes assigned a positive degree (ie, 1+, 2+).
quantitative – a characterization applied to laboratory tests that give results expressing a numerical
amount or concentration of an analyte in a specimen; NOTE 1: It is usually compared to an accredited
recognized standard; NOTE 2: This is in contrast to qualitative tests.
reference interval (RI) – interval between, and including, the lower reference limit to the upper
reference limit of the reference population (ISO/TR 18112-114); NOTE: For example, the RI for fasting
glucose measurement in plasma is 3.6 to 6.1 mmol/L.
reproducibility (measurement) – measurement precision (closeness of agreement between indications or

measured quantity values obtained by replicate measurements on the same or similar objects under
specified conditions) under reproducibility conditions of measurement (condition of measurement, out of
a set of conditions that includes different locations, operators, measuring systems, and replicate
measurements on the same or similar objects) (ISO/IEC Guide 99). 9
room temperature – the temperature prevailing in a working area; controlled room temperature – a
temperature maintained thermostatically that encompasses the usual and customary working environment
of approximately 18 to 25 °C; NOTE: Labeling can indicate storage at “controlled room temperature” or
“up to 25 °C.”
sample (patient) – one or more parts taken from a system and intended to provide information on the
system, often to serve as a basis for decision on the system or its production (ISO 15189)15; NOTE: A
sample is prepared from the patient specimen and used to obtain information by means of a specific
laboratory test.
sandwich immunoassay//two-site immunometric assay – a capture antibody, specific for the antigen, is
immobilized onto a solid phase, such as a polystyrene test tube or a microtiter plate well. The detecting
antibody binding the same or a different epitope of the antigen is labeled with an enzyme or other reporter
that is used as a signal-generating system. This can be used as a homogeneous assay.
sensitivity (of a measuring system) – quotient of the change in an indication of a measuring system and
the corresponding change in a value of a quantity being measured (ISO/IEC Guide 99)9; NOTE: From
predictive value theory, the proportion of subjects with a condition who have a positive test.
specificity (of a measuring system) – the ability of the test to correctly identify the absence of the
disease at a particular decision threshold (ie, a negative result and identification of patients who do not
have a disease); NOTE: From predictive value theory, the proportion of subjects who do not have a
condition who have a negative test.
specimen (patient) – the discrete portion of a body fluid or tissue taken for examination, study, or
analysis of one or more quantities or characteristics, to determine the character of the whole; NOTE: For
plasma-based coagulation testing, the specimen would be anticoagulated whole blood or plasma.
spiral (or helical) cone beam computed tomography – a type of three dimensional computed
tomography (CT) in which an X-ray source describes a helical trajectory relative to the object while a two
dimensional array of detectors measures the transmitted radiation on part of a cone of rays emanating
from the source; NOTE: More recently, the term CT pulmonary angiography has also been used
synonymously.
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threshold – an analyte concentration above or below which a specific action is indicated; NOTE: In the
case of D-dimer, the concentration below which venous thromboembolism can be excluded in the
appropriate clinical setting.
venous thromboembolism (VTE) – a thrombus (blood clot) or embolus in the venous circulation.
4.3 Abbreviations and Acronyms
CI confidence interval
CT computed tomography
CV coefficient of variation
DDU D-dimer unit
DIC disseminated intravascular coagulation
DVT deep vein/venous thrombosis
EDTA ethylenediaminetetraacetic acid
ELISA enzyme-linked immunosorbent assay
ER emergency room
FDA US Food and Drug Administration
FDP fibrin(ogen) degradation product
FEU fibrinogen equivalent unit
IRP international reference preparation
ISO International Organization for Standardization
NPV negative predictive value
PE pulmonary embolism/embolus
POC point-of-care
PPV positive predictive value
PTP pretest probability
QC quality control
RI reference interval
UEDVT upper extremity deep vein/venous thrombosis
VTE venous thromboembolism
5 Overview of D-dimer Testing

In 1972, it was predicted that the detection of crosslinked fibrin derivatives, a marker of in vivo fibrin
formation and degradation, “might prove useful as a diagnostic tool,” although utility was limited because
their detection by polyacrylamide gel electrophoresis was not suitable for routine clinical use.16 The
following year, a unique degradation fragment was identified after subjecting human fibrin to the
hydrolytic action of plasmin.17 This unique fibrin fragment was named D-dimer.18 A decade later, a solid-
phase enzyme immunoassay using monoclonal antibodies against D-dimer with a sensitivity to low
concentrations of D-dimer in plasma was reported. Soon after, two separate groups reported increased
concentrations of D-dimer in the plasma of patients with PE, DVT, DIC, and arterial thrombosis.19,20 Over
the next decade, multiple studies were published confirming that D-dimer concentrations are uniformly
elevated in individuals suffering from acute VTE and that concentrations below a certain established
threshold may be useful in the exclusion of this condition.21
5.1 Venous Thromboembolism Background
VTE is a condition that can have potentially serious and even life-threatening consequences. The most
common forms of venous thromboembolic disease are lower extremity DVT and PE. The yearly
incidence of VTE in the general population is reported as 1 in 1000 individuals, with an increasing
incidence of 1 per 100 in individuals 75 years of age or older. The incidence tends to be lower in younger
individuals such that the risk of VTE is approximately 1 in 10 000 in a healthy female population aged 15
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to 49 years.22 Therapy for thromboembolic disease using anticoagulation with a vitamin K antagonist
carries a 2% risk of major hemorrhage and a 0.5% to 1% mortality rate over a three- to six-month
treatment regimen. Therefore, anticoagulant therapy should be given only to those patients with objective
evidence of disease.
The diagnosis of VTE based on clinical findings alone is unreliable, and, therefore, objective testing is
necessary before a diagnosis of VTE is made. Both the increased availability of noninvasive imaging
techniques along with reduced tolerance for diagnostic uncertainty (possibly due to medical-legal risk)
have resulted in the referral of increasing numbers of patients with suspected VTE for diagnostic imaging
studies. Consequently, the proportion of patients with suspected VTE who in fact have a thrombus
confirmed by objective testing has fallen to between 15% and 25%, whereas prevalence as low as 5%
have been reported.23 Although venous ultrasound to evaluate for DVT is a noninvasive procedure, there
is a major cost and workload implication in sending a large proportion of patients for study (up to 90%)
that do not have venous thrombosis. Imaging analysis for PE also has its limitations. Ventilation-
perfusion lung scan, one of the most common methods employed in this evaluation, is fully diagnostic in
only 20% to 30% of the cases, and pulmonary angiography, the “gold standard” test for detection of PE,
carries a 0.5% mortality rate and a 1% rate of major complications. Spiral computed tomography (CT)
was more recently introduced in the evaluation of PE and has been shown to safely replace both
ventilation-perfusion scan and pulmonary angiography to safely establish or exclude PE.24 This testing is
costly, and for this reason, a sensitive, inexpensive, noninvasive screening test is highly desirable. D-
dimer measurement, in conjunction with a standardized assessment of PTP, is successfully used to
exclude VTE without the need for diagnostic imaging, provided the threshold for the D-dimer assay has
been objectively established and validated for this purpose.
5.2 D-dimer Measurement
D-dimer refers to a mixture of crosslinked fibrin(ogen) degradation products (FDPs) that each contains one
or several D-D domain motifs. In order to use laboratory assays that measure products related to the
formation and dissolution of blood clots to exclude the presence of venous thrombosis, they must be
sufficiently sensitive. When using assays with a high negative predictive value (NPV), D-dimer
concentrations below a predefined threshold imply that too little thrombus is present to be clinically
significant. In the evaluation of venous thrombosis, the D-dimer assay has its greatest utility when used for
its NPV (true negatives/all negatives). That is, a negative D-dimer (concentration below a predetermined
threshold) excludes the presence of a clinically relevant VTE. Controlled studies show that a negative,
sensitive D-dimer assay enables the exclusion of thrombotic events in up to 30% of patients. Therefore, the
potential to safely exclude a large portion of patients from further diagnostic evaluation is possible. As the
prevalence of VTE in the population tested rises, the probability of a false-negative test also rises; therefore,
do not use a quantitative D-dimer assay alone to exclude VTE in patients with high PTP.
As a product of fibrin dissolution, D-dimer concentrations are elevated in many other pathological
conditions, eg, DIC, sepsis, malignancy, inflammation, and following trauma or surgery. Concentrations
tend to increase with age and are generally elevated in otherwise normal pregnancy. This guideline
addresses the use of quantitative D-dimer only in the exclusion of venous thromboembolic disease in which
the PTP is low or intermediate (VTE unlikely). Selected studies have indicated the potential value of D-
dimer studies in patients who have high PTP or high PTP with negative imaging.25,26 However, guidelines of
the British Thoracic Society and British Committee for Standards in Haematology have recommended that
D-dimer testing be limited to patients with PTP that is low or intermediate.27,28
A number of D-dimer methods are rapid, simple, and inexpensive (relative to the cost of imaging), with a
high sensitivity and high NPV for VTE. A reliable threshold value, which has been validated to have a
high NPV for VTE when results fall below that threshold, is essential for interpreting the test result.
However, despite the widespread use of D-dimer assays, many clinicians are confused by the variation in
units, numerical results, and threshold values used by the different assays.
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5.3 Problems With D-dimer Measurement
The fibrin fragment D-dimer is the smallest crosslinked degradation product of crosslinked fibrin.
However, monoclonal antibodies raised against fragment D-dimer also react with crosslinked fibrin and a
variety of crosslinked FDPs. Consequently, D-dimer is not a uniform analyte. Although fragment D-
dimer is the minimal structure detected by D-dimer antigen-specific monoclonal antibodies, the
monoclonal antibodies detect a “soup” of crosslinked FDPs containing this antigen. Clinical samples
typically contain varying amounts of D-dimer fragments and high-molecular-weight crosslinked FDPs.
Different antibodies have different specificities to these products with some preferentially binding to
high-molecular-weight crosslinked products, whereas others preferentially bind to low-molecular-weight
fragments. In addition to the antibody selection, the detection principle (turbidimetric/nephelometric,
enzyme-linked immunosorbent assay [ELISA], or others) is relevant for different sensitivities to D-dimer
products. Cross-reactivity with FDPs may be a serious limitation of certain assays,29 but this interference
has not caused a problem in the exclusion of VTE.
The molecular weight of a D-dimer fragment is approximately one half of the molecular weight of the
fibrinogen molecule. Two methods are now used by manufacturers to express the weight of the molecule,
either the D-dimer unit (DDU) expressing the weight of the fragment itself or the fibrinogen equivalent
unit (FEU) expressing the weight of the fragment in terms of the fibrinogen (fibrin) molecule from which
it was derived. Thus, 2 ng/mL FEU is equivalent to 1 ng/mL DDU. This can create confusion when the
type of units being used is not clear to the clinician. Finally, the magnitude of the units is expressed
differently by different manufacturers, eg, ng/mL, µg/mL, µg/L. This variability of the type and
magnitude of the units is a source of confusion for both laboratories and clinicians.
5.4 Harmonization vs Standardization
The lack of a D-dimer standard is a problem. One of the major causes of poor standardization of D-dimer
assays is the absence of an international reference preparation (IRP). Achieving this IRP is complicated
by the marked variety of D-D–containing molecules in biological material that cannot be approximated by
mixtures of (purified) fibrin fragments. Manufacturers generally select calibration materials to reflect the
requirements of their assays, rather than to achieve comparability among D-dimer assays. Calibration
materials may include purified D-dimer fragment; semipurified high-molecular-weight crosslinked fibrin
products, prepared from purified fibrin; or pooled plasma samples. The calibrator matrix may be a buffer,
or a buffer with added plasma, serum, fibrinogen, or other proteins of human or animal origin.
Initial attempts to standardize D-dimer assays used purified D-dimer fragments as a calibrator. This
approach was not viable because different reagent kits use different antibodies. Not all antibodies
appeared to recognize the purified D-dimer fragment. Also, some latex agglutination assays were unable
to crosslink using these small fragments.30 During the initial attempts, it was believed that standardization
of D-dimer assays was not feasible, so attempts were made to harmonize the assays using panels of
plasma samples from patients with a range of conditions in which the hemostatic balance was
disturbed.29,30 These studies demonstrated that clinical samples showed less interassay variation than
purified FDPs, reflecting the heterogeneity of crosslinked products in clinical samples. The best
agreement among methods was found when pooled plasma samples from patients with high D-dimer
concentrations were used. Using this approach, the ratio between the consensus value of all D-dimer
methods and the value of a particular method for pooled plasma was used to create a conversion factor.
This harmonization resulted in good agreement on average, but important discrepancies remained for
individual samples, in particular at higher concentrations.31
Meijer et al.32 prepared a set of five plasma samples with increasing D-dimer concentrations by mixing
plasma pooled from patients with high D-dimer concentrations with pooled normal plasma. This set of
plasmas was distributed to more than 500 laboratories, and the data were used to calculate a reference line
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from overall median D-dimer values at each concentration and a method-specific regression line.
Harmonization was then achieved for the methods included in this exercise by transforming a method-
specific regression line to the reference line. Between-method variation was markedly reduced using this
model, and the authors suggested that manufacturers could apply the harmonization procedure to their
calibrators. Although this approach has much to recommend it, some problems remain: only seven assays,
all produced in Europe, were harmonized, and the addition of new assays to the scheme would be
expected to cause a shift in the reference line. Also, differences among the composition of the
manufacturers’ calibrators remain, and it may be difficult on an ongoing basis to obtain the volume of
plasma required from patients with pathological conditions needed to create such pools for use.
Given these constraints, it is suggested that in vitro preparations containing a similar range of crosslinked
FDPs to that found in clinical samples be used as a reference material.29 The ideal reference material would
have a high concentration of D-dimer and would be virus free or virally inactivated. The “true” D-dimer
content would be ascertained by preparing a terminal plasmin digest so that all D-dimer antigen contained in
high-molecular-weight products would be broken down to fragment D-dimer. When a suitable IRP is
produced and the manufacturers adopt its use, standardization of D-dimer assays may be possible. Until that
time, harmonization has the greatest potential as a practical alternative to the bewildering array of units and
threshold values currently in use.33
6 Patient Evaluation and Pretest Probability

No single test, including the D-dimer assay, has sufficiently high sensitivity and specificity to use alone in
the diagnosis or exclusion of patients with suspected venous thrombosis. However, the D-dimer test is a
powerful adjunct when used with other diagnostic or clinical assessment modalities in the evaluation of
VTE.
Venous thromboembolic disease cannot be diagnosed with certainty based on clinical evaluation alone.
This is largely because the clinical signs and symptoms of both DVT and PE are insensitive and
nonspecific. Although VTE cannot be accurately diagnosed on clinical grounds, there is good evidence
that clinicians can correctly and reproducibly classify patients into categories of either “VTE likely” or
“VTE unlikely” or “low,” “intermediate,” and “high probability of VTE.” Accurate determination of
disease likelihood (PTP) has been facilitated by standardized assessments that use scoring systems based
on criteria and risk factors derived from large databases (see Tables 1 and 2). Sets of standardized
prediction rules have been proposed in the evaluation of the clinical probability of both DVT (eg, Wells34)
and PE (eg, Wells and colleagues35 and the Geneva group36). These criteria have been validated in clinical
trials and have also been reliable when used by other clinical groups.
Using clinical assessment decision rules to determine the likelihood that a patient is experiencing VTE, in
combination with D-dimer testing, can improve the diagnostic testing strategy for the evaluation of
thromboembolism over the use of either assessment alone. In this setting, the quantitative D-dimer assay has
its greatest utility for its NPV when applied to patients classified as “unlikely” or “low-intermediate” clinical
probability. In these patients, depending on the NPV of the D-dimer assay used, a D-dimer result below the
exclusion threshold value suggests strongly against the presence of VTE, such that the diagnosis can be
confidently excluded and no further diagnostic procedures for VTE (imaging) need be performed. When
clinical probability of VTE is high (or “likely”), D-dimer testing is not informative and should not be
executed. In this instance, the appropriate diagnostic imaging tests, such as lower extremity ultrasound,
ventilation perfusion scanning, or spiral CT should be undertaken. When clinical probability falls in the low or
intermediate (unlikely) range, the ability to exclude VTE depends on the NPV of the D-dimer assay used.
When the D-dimer threshold is set too low, even otherwise normal, healthy individuals cannot be excluded.
When D-dimer assays are of limited sensitivity or the threshold is set too high, the potential for missing
clinically relevant thromboses exists. (Refer to Section 9 for additional information on determining
threshold.)
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Table 1. Wells Simplified Clinical Model for Assessment of DVT*,34

Clinical Variable Score
Active cancer (treatment ongoing, within previous six months, or palliative) 1
Paralysis, paresis, or recent plaster immobilization of the lower extremities 1
Recently bedridden for three days or more, or major surgery within the previous 12 weeks 1
requiring general or regional anesthesia
Localized tenderness along the distribution of the deep venous system 1
Entire leg swollen 1
Calf swelling at least 3 cm larger than that on the asymptomatic leg (measured 10 cm below 1
the tibial tuberosity)
Pitting edema confined to the symptomatic leg 1
Collateral superficial veins (nonvaricose) 1
Previously documented DVT 1
Alternative diagnosis at least as likely as DVT −2
* ≥2, probability of DVT is “likely.” ≤1, probability for DVT is “unlikely.” Alternatively, <1 is low
probability, intermediate is 1 or 2, and high is >2. D-dimer testing to be performed for exclusion of DVT
only if Wells Score is <2.
From Wells PS. Integrated strategies for the diagnosis of venous thromboembolism. J Thromb Haemost.
2007;5(suppl 1):41-50. Reproduced with permission of John Wiley & Sons, Ltd.
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Table 2. Predicting Probability of PE: Comparison of Geneva and Wells Scores*,†,37

Revised Geneva Score30 Wells Score35
Variable Points Variable Points
Pain on lower limb deep venous 4 Clinical signs and symptoms of DVT 3
palpation and unilateral edema (minimum of leg swelling and pain with
palpation of the deep veins)
Previous DVT or PE 3 Previous DVT/PE 1.5
Surgery (under general anesthesia) 2 Immobilization or surgery in the 1.5
or fracture (of the lower limbs) previous four weeks
within one month
Active malignant condition (solid or 2 Malignancy (on treatment, treated in the 1
hematological, currently active or last six months or palliative)
considered cured <1 year)
Hemoptysis 2 Hemoptysis 1
Heart rate 75–94 beats per min–1 3 Heart rate greater than 100 1.5
Heart rate >94 beats per min–1 5 An alternative diagnosis is less likely 3
than PE
Unilateral lower limb pain 3
Age >65 years 1
Clinical Probability Clinical Probability

Low 0–3 total Low <2 total
Intermediate 4–10 total Intermediate 2–6 total
High >10 total High >6 total
*D-dimer testing to be performed for exclusion of PE only if the Geneva Score is <10.
†
D-dimer testing to be performed for exclusion of PE only if the Wells Score is <6.
Adapted from Wells PS, Anderson DR, Rodger M, et al. Derivation of a simple clinical model to
categorize patient’s probability of pulmonary embolism: increasing the models utility with the
SimpliRED D-dimer. Thromb Haemost. 2000;83(3):416-420; and Ten Cate-Hoek AJ, Prins MH.
Management studies using a combination of D-dimer test result and clinical probability to rule out venous
thromboembolism: a systematic review. J Thromb Haemost. 2005;3(11):2465-2470. Reproduced with
permission of John Wiley & Sons, Ltd.
6.1 Limitations Related to Patient Conditions or Therapies
Several patient conditions and therapies influence D-dimer concentrations independent of the occurrence
of a thrombosis. The following conditions are associated with an increase in D-dimer concentrations, even
in the absence of VTE:
• Fibrinolytic therapy within the previous seven days

• Trauma or surgery within the previous four weeks
• Large hematoma
• Disseminated malignancies
• DIC
• Sepsis, severe infections, pneumonia
• Liver cirrhosis
• Pregnancy38
• Atherosclerotic vascular disease
• Sickle cell disease
• Advanced age (>60 years)
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There are some situations in which a D-dimer value may fall below an established threshold in the
presence of a venous thrombosis. This may occur in individuals who start on therapeutic doses of rapid
acting anticoagulants (eg, unfractionated heparin, low-molecular-weight heparin, pentasaccharide, and
other direct thrombin and Xa inhibitors) 24 hours or more before the D-dimer is measured. Anticoagulant
therapy may lead to reduced concentrations, and, therefore, a false-negative D-dimer in the presence of
VTE. Thus, for exclusion of venous thrombosis or PE, the analyte D-dimer should not be used as a
diagnostic aid in patients on anticoagulant therapy.39 Another situation in which the D-dimer value may
be below threshold in the presence of thrombosis is if the clot is small and of insufficient size to raise D-
dimer values above the threshold. This is more likely to occur if the clot develops distal to the knee,
where there is typically a trifurcation of the deep vein of the leg, significantly reducing the venous caliber.
D-dimer should not be used to exclude lower extremity DVTs if the suspected thrombus is distal to the
knee. In addition, small (subsegmental) PEs may also fail to raise the D-dimer concentration above the
threshold for exclusion of PE.40 For this reason, D-dimer may not be appropriate to exclude upper
extremity deep vein/venous thrombosis (UEDVT) because D-dimer values appear to be significantly
lower in UEDVT compared with lower extremity DVT.41 D-dimer concentrations may also fall below
threshold if the elapsed time between the onset of thrombosis and the measurement of D-dimer is of
sufficient duration such that D-dimer has been cleared from the circulation. The duration of time needed
between presentation and concentration of D-dimer falling below the established threshold depends on
the initial size of the clot. A smaller clot may be associated with D-dimer concentrations that fall below
threshold in a shorter period of time than would occur if the DVT were larger. In the rare situation of a
deficiency of tissue plasminogen activator, D-dimer concentrations may fall below the threshold value
despite the presence of clot. The possibility also exists that the diagnosis of a clot in the presence of a
negative D-dimer represents a false-positive imaging study. Finally, the use of a quantitative D-dimer for
the exclusion of VTE cannot be recommended in children. In one published report, 15% of children with
PE had normal D-dimer values. Furthermore, for many reasons, PTP scores such as the Wells simplified
probability score for PE do not perform as well in children as in adults and, therefore, are not likely useful
tools in this population.42,43 The D-dimer is elevated in nearly 80% of patients who are hospitalized, which
is the reason behind the general recommendation that the D-dimer test not be used in the evaluation of
these patients for VTE. The conditions associated with this high rate of positive tests are included in the
list earlier in this section. However, D-dimer may be useful in hospitalized patients who do not have
comorbid conditions that cause D-dimer elevation, such as patients in rehabilitation.44,45 There are some
unique points regarding the use of D-dimer for exclusion of VTE in pregnancy. The diagnosis of VTE in
pregnancy remains problematic and the role of D-dimer continues to be debated.38,46,47 Because the D-
dimer rises as a result of pregnancy, it can be used only with great care. Because guidance for D-dimer
use in this clinical setting is limited, the laboratory would need to develop the threshold for its use based
on local criteria. In general, the use of the test for exclusion of VTE in pregnancy is not recommended.
7 Specimen Collection and Processing

Closely monitor variables in the preexamination phase of D-dimer testing to ensure that results accurately
reflect the true condition of the patient. Many of these variables are addressed below.
7.1 Patient Variables/Preparation Before Collection
The concentration of measurable D-dimer in plasma depends on the size and age of the thrombus. D-
dimer elevations were detectable when tested in patients just two hours after inducing a thrombus.48 The
approximate half-life of D-dimer is seven hours.49 This information may be useful in interpreting
sequential quantitative (or semiquantitative) results in a patient over a given time period. The half-life of
D-dimer is also pertinent to sensitivity of the assay for the exclusion of VTE. Sensitivity for ruling out
VTE decreases as the age of the clot increases. Individuals who present for D-dimer testing weeks to
months after symptoms of VTE appear are significantly more likely to have a D-dimer concentration
below a given threshold than if they had presented at the onset of signs or symptoms.
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Sensitivity of D-dimer for the exclusion of VTE is decreased in those patients who are tested while taking
oral anticoagulant therapy. In this instance, a false-negative D-dimer result is more likely.50
Following major surgery, D-dimers increase, peaking on day seven, and thereafter clearing at a rate of
approximately 6% per day. D-dimers remain elevated for four weeks or longer, depending on the
surgery.51
7.2 Specimen Collection
Samples for D-dimer testing are collected using either evacuated tube systems or syringes. Depending on
the assay method used, either whole blood or plasma samples are appropriate for testing. D-dimer assays
contain antibodies that do not react with native fibrinogen, allowing plasma to serve as a suitable
specimen type.
The majority of plasma-based commercially available assays require sodium citrate plasma. For practical
reasons, it is generally recommended that CLSI guideline H2152 be followed for specimen collection
because it addresses in detail samples with an elevated hematocrit, the effect of underfilling or overfilling
evacuated collection tubes, the sodium citrate concentration recommended, and the proper procedure for
obtaining samples through a vascular access device. Collect venous blood as recommended by the
manufacturer, usually into 109 mmol/L (3.2%) or 129 mmol/L (3.8%) of trisodium citrate, at a ratio of
nine parts blood to one part anticoagulant. (The recommended concentration of trisodium citrate is 109
mmol/L.)
In some D-dimer assays, results are not influenced by the anticoagulant used for blood collection, and
sodium citrate, ethylenediaminetetraacetic acid (EDTA), or heparinized plasma can be used. Depending
on the assay system, especially if a point-of-care (POC) analyzer is employed, EDTA samples can be
used as whole blood or as EDTA plasma. Some POC-type analyzers require heparinized whole blood as
the only suitable sample type. Always follow manufacturers’ instructions for appropriate sample type and
usage.
When collecting a sample for D-dimer testing, the use of a discard tube is not necessary.53 Avoid stasis or
trauma during phlebotomy because these conditions may lead to spuriously elevated D-dimer
concentrations. If collecting the sample using an evacuated tube system, adequately fill the evacuated
tube. The presence of a clot within the tube is a cause for sample rejection.
7.3 Specimen Processing, Transport, and Storage
In general, follow published guidelines for processing, transport, and storage of hemostasis samples (see
CLSI document H21).52 Follow the manufacturer’s specific instructions, and locally validate any deviation.
D-dimer is a stable measurand that may tolerate storage times of up to 24 hours at room temperature or in
a refrigerator, and plasma samples may be stored for up to 24 months at −24 to −74 °C.54,55
Transport frozen plasma samples (prepared after centrifugation and aliquotting) on dry ice, using the
appropriate regional dangerous goods transport rules. Before testing, thaw frozen plasma samples in a 37 °C
water bath for approximately five minutes. The length of time samples are subjected to the heated water
bath depends on the frozen sample volume. Thoroughly mix samples before testing. Particulate matter
present in the thawed plasma sample may affect test performance. When provided, the manufacturer’s
procedures should be followed to address specimen processing, removal of particulates, transport, and
storage.
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7.4 Interferences
Lipemic or hemolyzed samples may cause interference, especially in quantitative, microparticle

agglutination assays. The amount of interference depends on the wavelength of light used, as well as the
severity of the lipemia and/or hemolysis. The manufacturer’s package insert should include interference
data when necessary. Refer to CLSI document EP0756 for additional information about the effects of
interfering substances. If deemed applicable, diluting the sample with an appropriate solution may
minimize the interference.
High levels of rheumatoid factor may cause interference in some microparticle agglutination
immunoassays. However, one commercially available D-dimer assay specifically states that no
interference from rheumatoid factor has been observed in that particular assay.57
Although it is rare, the presence of heterophile antibodies can interfere with commercial assays owing to
the animal origin of the monoclonal or polyclonal antibodies present in their respective reagent
formulations. This phenomenon can be seen with cryoglobulinemia.58 For these problem samples, an
alternative methodology that contains large amounts of nonimmune animal antibodies that minimize
interference from heterophilic antibodies may be used. (Refer to CLSI document I/LA3059 for additional
information about interfering antibodies.) Commercially available products designed to pretreat problem
samples can remove heterophile antibodies before assay. These products are more commonly used in the
chemistry laboratory but may prove a suitable option when a patient presents with heterophile antibodies.
Use of such agents may be incompatible with certain reagents, and, therefore, validation is necessary.
8 Assay Methodologies
In choosing a D-dimer method to use for the exclusion of VTE, it is important to evaluate the sensitivity
and NPV of the assay. Additional criteria to consider when selecting kits or methods include:
• Qualitative vs quantitative
• Time to result
• Availability of the assay at all times (batching samples is associated with delay to result)
• Acceptable precision at or around the threshold
• Reporting unit of the assay: FEU vs DDU, nanograms per milliliter, micrograms per liter, micrograms
per milliliter, milligrams per liter
• Well-established diagnostic threshold in the reporting unit: manufacturer-established vs site-
established
• Intended use of the product: exclusionary claims vs aid in diagnosis
• Clinically safe, noninvasive, time- and cost-effective
8.1 Quantitative Sandwich Assays
Quantitative sandwich assays are categorized as ELISA and non-ELISA tests. ELISA tests used for the
determination of D-dimer are two-site immunometric assays (so-called sandwich ELISA).60 A capture
antibody specific for D-dimer is immobilized onto a solid phase, such as a polystyrene test tube or a
microtiter plate. The detecting antibody, binding the same or a different epitope of D-dimer, is labeled
with an enzyme and is used as a signal-generating system. Depending on the specificity of the detecting
antibody, the binding of the two antibodies, with the sample containing the antigen and consisting of one
or more dimerized D-domains, may be realized in one or two steps. In the case of a two-step method,
unreacted sample is removed by washing before the detecting antibody is added. Excess detecting
antibody is then removed by a second washing. Depending on the substrate used, a chromogenic,
fluorogenic, or chemiluminescent signal is produced and measured. The signal is proportional to the
amount of D-dimer present.
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ELISA assays are developed using several different variations based on the solid phases, antibodies, and
signal generation system. These methods are detailed in general references for immunoassays.61
For automated heterogeneous assays, many different solid phases are common, such as polystyrene tips,
beads, dendrimers, or microparticles in conjunction with membranes as with radial partition
immunoassays and magnetic particles with microparticle immunoassays. Dendrimers, microparticles, and
micron-sized magnetic particles are in suspension during the incubation period. Therefore, molecular
diffusion distances are reduced, thus accelerating the reaction kinetic. As a signal-generating system,
alkaline phosphatase with 4-methylumbelliferyl phosphate (enzyme-linked fluorescent assay)62 or
adamantyl dioxetane anion is used. Other systems apply acridinium esters, isoluminol, or sulfonamides,
generating a direct chemiluminescent signal without enzymatic reaction.
8.2 Quantitative Microparticle Agglutination
Microparticle agglutination is a homogeneous assay design frequently used for the determination of
D-dimer. Homogeneous immunoassays require only mixing of a sample and immunochemical reagents,
followed by detection. Immunochemical binding produces a physically detectable signal, which obviates
the need to separate bound from free label. The intensity of light scattering by microparticles depends on
their size; thus, the agglutination of microparticles caused by the immunochemical reaction changes the
amount of scattered light as the reaction proceeds. For quantitative D-dimer assays, uniform polystyrene
particles with diameters in the range of 0.10 to 0.5 µm are preferred. The particles are coated with one or
two specific antibodies. The agglutination is measured turbidimetrically or nephelometrically.
Turbidimetry measures the intensity of a beam of light transmitted through the sample, and nephelometry
measures the light that is scattered at an angle away from the initial direction of the beam. To reduce
interference due to particulate matter in the sample, a measurement of the change in absorbance after
sample addition is performed. The change in absorbance with time is proportional to the concentration of
D-dimer present. Most of these assays have a typical time of analysis of less than 10 minutes plus
centrifugation time, and they are available on automated analyzers and coagulometers63 of routine
laboratories and emergency rooms.
Microparticle agglutination is established using several different variations based on the particles and
antibodies used. These methods are detailed in general references for particle agglutination.64,65
8.3 Semiquantitative Microparticle Agglutination Methods
Among the older types of D-dimer assays are the manual semiquantitative latex agglutination methods.
Although these assays are useful for detecting larger circulating quantities of D-dimer as occurs in DIC,
thrombolysis, or other conditions, they are considered too insensitive for exclusion of VTE.21,66-68
These assays are typically performed by mixing the patient sample with antibody-coated latex beads on a
test card or slide. If D-dimer concentrations are significantly elevated, agglutination occurs, causing visible
clumping. Slides are viewed macroscopically by a technologist or other clinician using a direct light source
to determine if agglutination is present or absent. These assays have a high interobserver variability of end-
point determination.60,61 Currently published studies demonstrate clearly that it is inappropriate to use these
assays for the exclusion of VTE.21,66-68
8.4 Point-of-Care Testing
POC D-dimer assays have been considered by smaller laboratories when quantitative
methodologies/results are desired, but coagulation instrumentation with the capability of automated
quantitative D-dimer testing is lacking. These systems may also be advantageous in settings in which an
immediate result is desired with the shortest possible turnaround time. Stand-alone POC analyzers may
have different quality control (QC) requirements than the usual laboratory assay; for example, some
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systems provide “electronic” as well as liquid QC. Manufacturers’ recommendations for QC must be
strictly followed.
Several POC D-dimer assays are available. These assays are typically performed on whole blood or
plasma, depending on the assay.69 To date, the number of clinical studies documenting the performance
of these methods is relatively small, and in many of these studies, the sensitivity for VTE was <97%
and NPV was near or <97%, depending on the method and the threshold (see Section 9.1, Table 3 for
recommended assay sensitivity and NPV).70-86 At present, none of the POC methods have been cleared
by the US Food and Drug Administration (FDA) for exclusion of VTE.
8.5 Other Technologies
For cost-effective automation, the assay format should use as few washing and incubation steps as
possible and use the shortest incubation time, without impairing sensitivity or specificity. Consequently,
many different solid phases and signal-generating systems have been developed and are used for D-dimer
determination. Besides heterogeneous assays (see Section 8.1) and classic quantitative microparticle
agglutination (see Section 8.2), further homogeneous techniques have been developed. Because
homogeneous assays need no separation steps, the requirements for automation are minimal.
Homogeneous assays are typically based on the light scattering of microparticle agglutination, but also
more sophisticated signal-generating systems like fluorescence energy transfer immunoassays,
fluorescence resonance energy transfer immunoassays, and luminescence oxygen-channeling
immunoassays are, in principle, suitable for D-dimer determination and may be commercialized in the
near future. These methods are detailed in general references for immunoassays.58,61
The performance characteristics of assays based on these promising technologies are yet to be
determined.
9 Assay Characteristics, Reference Interval, and Threshold for Exclusion of

Venous Thromboembolism
9.1 Recommended Assay Characteristics
When applied correctly, specifically in conjunction with a PTP model as part of a clinical algorithm, the
use of the D-dimer assay can improve the efficiency of the evaluation of the patient suspected of having
clinically relevant VTE. Incorrectly excluding VTE would place the patient at high risk for added
morbidity or even mortality. To use the D-dimer assay for exclusion of VTE, the test must meet certain
characteristics to protect patient safety. There must be a high NPV and sensitivity, good reproducibility at
the level of the threshold for exclusion of VTE, and sufficient specificity to provide discrimination. A
quantitative D-dimer assay should not be used to exclude VTE in patients with high (likely) PTP.
The recommended predictive value characteristics at the threshold for the level of the exclusion of VTE in
patients with low/intermediate (unlikely) PTP for a D-dimer assay to use for the exclusion of VTE are
included in Table 3. In most cases, the manufacturer of the assay provides the NPV and its confidence
interval (CI). It is recommended that manufacturers provide not only the NPVs of their tests, but also
references to generally accessible publications of these data, eg, the peer-reviewed literature or publicly
accessible government/regulator or accrediting agency databases. Imprecision of the assay is reflected in
the coefficient of variation (CV). Many D-dimer assays report a within-laboratory, between-run CV of
7.5% or less (eg, at this CV, the 95% CI for an assay value of 500 ng/mL ranges from 425–575 ng/mL;
for an assay value of 0.5 µg/mL, it ranges from 0.43–0.58 µg/mL; for an assay value of 1.0 mg/L, it
ranges from 0.85–1.2 mg/L). This CV should be achieved by quantitative D-dimer assays. Guidance for
determining assay imprecision for manufacturers is found in CLSI document EP05.87 Guidance for
laboratories to validate imprecision in their own locations is found in CLSI document EP15.88 The
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regulatory requirements in different countries are not identical. Those of the FDA of the United States are
detailed in the appendix. In the United Kingdom, the requirements are slightly more rigorous, requiring
that the NPV reach the level 98%. Recommended assay characteristics are shown in Table 3.28
Table 3. Recommended Assay Characteristics When Testing Patients With Low or Intermediate
PTP of VTE
NPV ≥98%
The 95% lower limit of the one-sided CI of the NPV ≥95%
Sensitivity ≥97%
The 95% lower limit of the one-sided CI of the sensitivity ≥90%
9.2 Reference Interval
Assays that have both an RI and a threshold for the exclusion of VTE can lead to confusion for clinicians
because the threshold for exclusion of VTE may be the same as, higher than, or less than the upper limit
of the RI. When reporting the value of the D-dimer performed for the exclusion of VTE, it is best if only
the threshold for exclusion of VTE is reported to avoid confusion, particularly if the threshold and the
upper limit of the RI are not the same. The RI is not useful when using the D-dimer for the exclusion of
VTE. In many clinical laboratories, the reason for performance of the D-dimer is not known at the time of
testing. In that case, it is recommended that both the RI and the threshold for exclusion of VTE are
included with the report.
D-dimer is elevated in some apparently healthy groups of patients, including patients who are pregnant,
patients of advanced age89 (>60 years), and those in the perinatal period. In these patients, using the D-
dimer to exclude VTE is of limited value.
When using the D-dimer to evaluate other clinical conditions, the RI is important and must be reported
with the result. For full information on determination of the RI, see CLSI document C28.90
9.3 Establishing the Threshold for Venous Thromboembolism Exclusion: Comparison to

Clinical and Imaging Studies
Clinical and imaging criteria for the diagnosis of DVT and PE have been described. The development of
the threshold below which VTE can be excluded requires that testing be performed in a population of
patients suspected of having VTE in whom the diagnosis has been established or excluded using imaging
techniques. To fulfill criteria of no evidence of VTE, it is also necessary to re-evaluate the patient after
three months to confirm the negative diagnosis. This “final diagnosis” becomes the “truth” in predictive
value theory analysis required for the following procedure. In addition, data regarding the use of both
clinical criteria and D-dimer for exclusion are confined to exclusion of PE and proximal DVT. They
cannot be applied (and may be misleading) in the evaluation of thrombotic events in other locations,
including UEDVT and arterial thrombosis. Patients with distal DVT may have a D-dimer below the
threshold for exclusion of VTE in 35% of cases; therefore, these results cannot be used to avoid
ultrasound evaluation. Most frequently, patients with distal DVT may require imaging evaluation. D-
dimer testing used in concert with lower extremity ultrasound evaluation may be helpful.
When the D-dimer test is used for the purpose of exclusion of VTE, a critical issue is the determination of
the threshold below which the clinician can confidently exclude VTE. Therefore, it is important to
demonstrate that the performance of the test at the threshold meets predetermined criteria that will ensure
its desired performance and patient safety.
The number of cases that must be studied to validate the threshold is substantial and beyond the
capabilities of the majority of laboratories. In 2009, Steinberg and colleagues91 described a procedure for
sample size estimation that can provide guidance should a facility desire to determine and validate the
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threshold. The sample size depends on the prevalence of VTE in the population studied, the NPV desired,
and the sensitivity and specificity of the test. Higher prevalence in the population tested increases the
sample size because of the accompanying increase in the number of false-negative tests. If the patients
with clinically high PTP (likely PTP) are excluded and only those with low and intermediate PTP
(unlikely PTP) are tested, the prevalence will be less than 10%, reducing the number of subjects required
for the study. However, even with this pretest screening, hundreds of subjects are required.
Additionally, regulatory agencies may require that the threshold for exclusion of DVT and PE is
determined separately and may require that the data are collected at multiple institutions.
Whether using the Bayesian approach (random sample of true-positive and true-negative subjects as
described by Steinberg86) or the random sample of test-negative subjects approach (as described by
Fleiss),92 estimating the required sample size for establishing NPV depends on a number of variables and
the characteristics of the test being evaluated. If one demands, in addition to high sensitivity (0.97) and
high NPV (0.98), a specificity of 0.40 or higher and power that exceeds 0.80, then the sample size is very
high, as great as 2500 to 3000. However, even though the requirement for NPV may be 0.98, tests may
have the threshold for exclusion set very low in order to ensure the minimum false-negative test
(accepting a lower specificity), such that the actual NPV is >0.99. Such parameters lead to a requirement
for a lower sample size. Regardless, determining the threshold with sufficient power and high confidence
requires the testing of a minimum of 200 to 300 cases.88,91
Finally, if the population tested is selected from archived, frozen samples, then a study of frozen and fresh
samples must be performed to demonstrate that the concentration of D-dimer determined by the method is
not affected by freezing and thawing the specimen. Compare values of original fresh samples with values
of frozen samples. When the new assay is compared with a clinically validated D-dimer assay, another
approach is to use “fresh” plasma samples from the daily routine and measure them together with the
precision (frozen) samples using the precision experiment found in CLSI document EP05.87 By this
approach, an adequate number of samples can be compared to detect uncommon outliers between assays.
Such an approach is recommended when a comparison and a tested method show a high correlation.
9.4 Methods That a Laboratory May Use to Determine the Threshold for Exclusion of
Venous Thromboembolism
If the D-dimer is used clinically for exclusion of VTE, the laboratory or clinical setting must determine
the threshold that will be used for the exclusion of VTE. This determination is accomplished in one of the
following ways.
The threshold may be determined by the manufacturer and the value may be cleared by a regulatory
agency for use with the method. Such information is published in the package insert of the reagents, and,
provided the same method is performed, this threshold is used. This is the best option. Laboratories in the
United States as well as some other locations internationally should be aware that only those assays with
an intended use stating that the assay can be used to rule out PE or DVT should be used as such. Other
options listed below are for use only in settings in which the manufacturer’s validation is not available,
and should be exercised with care.
The threshold can be derived from the peer-reviewed literature. The laboratory must use the same method
that is reported in the literature. Careful evaluation of the study used is necessary to ensure that
appropriate numbers of patients are included and that the statistical power of the study will validate the
threshold reported.
The threshold may be determined by another laboratory or group of laboratories, but the value is not
published. In this case, the laboratory must know the source of the data and have a summary of the data
used to determine the threshold including, but not limited to, the number of patients with and without
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thrombosis studied, the NPV, the sensitivity, the specificity, and the power of the determination. The
results of such a study should meet or exceed the statistical power of the study used by the manufacturer
to determine the threshold. In addition, the laboratory must use the same method as that used in the study.
Although the laboratory may determine the threshold using data collected in its own laboratory, this is
difficult because of the number of cases that must be studied to achieve appropriate statistical power.
Depending on the location, some of the methods described earlier may not be acceptable to regulatory and
accreditation agencies. Regardless, the recommended method is to use the threshold provided by the
manufacturer, if available.
There may be settings in which the laboratory system may have more than one site for performing the
D-dimer. With some analytes, correlation studies (harmonization) are used for validating the test in the
second site. This is not recommended for the D-dimer. As is apparent from the above-mentioned
information, there is considerable variability among methods, and efforts at harmonization may be
misleading. A possible exception is the situation in which the two sites use the same method (same
instrument, reagent, and lot number of the reagent). Harmonization may be an adequate method in this
setting. If the method is the same but lot numbers of reagent are different, variability may be higher and
harmonization more difficult or inadequate.
Owing to the complex nature of D-dimer, the data scatter in method comparisons is generally larger than
in automated immunoassays for typical monomeric antigens. Besides the different antibodies, in the
technique used, the liquid handling and the composition of reagent and dilution buffers may influence the
scatter significantly. Despite these constraints, several assays using different antibodies and different
methodologies correlate relatively well.93
9.5 Satisfying Regulatory Requirements
Manufacturers apply to regulatory agencies for clearance of a new method or for clearance of new
functionality of methods that have already been cleared. When applying to the FDA, they may seek either
an “exclusionary” claim or an “aid in diagnosis” claim for these assays (see Table A1 in the appendix).
Items to consider regarding such applications are included in Sections 9.1 and 9.3, and in the appendix.
The detailed requirements are unique to the regulatory agency to which the manufacturer applies, and the
manufacturer is referred to the agency for those detailed protocols. An example of the differences in data
required to support specific indications for use is found in Table A1 in the appendix.
There are many circumstances in which FDA approval or clearance has application beyond the United
States. For example, once a product is FDA approved or cleared, the manufacturer can request a copy of
the “Certificate to Foreign Government” from the FDA, which certifies that the product can be legally
marketed in the United States. This document serves as sufficient permission for many countries to
market the product. China’s State Food and Drug Administration will not begin the regulatory process
without evidence that the product has already been FDA approved, FDA cleared, or CE marked.
10 Interpretation of Results
D-dimer is one of the most widely used markers of in vivo clotting activation, mainly due to its
importance in excluding VTE in symptomatic patients. Any patient for whom acute VTE is suspected
requires rapid clinical assessment and objective testing to achieve an accurate diagnosis.
Patients presenting with symptoms consistent with VTE (symptoms for DVT may include leg pain,
tenderness, swelling, discoloration, and/or edema, whereas symptoms for PE may include labored
breathing, coughing, and lung-related chest pain) are first evaluated with a PTP scoring method such as
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the Wells scheme37,94-98 or the revised Geneva score.95 (See Section 6.) The PTP score is calculated from a
series of risk factors and patient symptoms defined in one of these pretest evaluations to quantify the
patient’s risk for VTE. Thus, patients whose PTP indicates low or intermediate risk (unlikely risk) should
be considered for D-dimer testing, whereas patients whose PTP indicates high risk (likely risk) cannot
have VTE excluded with sufficient sensitivity using the D-dimer assay and should be considered for an
imaging procedure.
The risk category to which the patient is assigned dictates the follow-up testing to perform. For example,
patients deemed to be at low risk typically undergo noninvasive follow-up testing, whereas patients
deemed to be at high risk may undergo more invasive imaging procedures. D-dimer is considered a
noninvasive method. As the PTP score increases, the probability that a positive D-dimer test is a true
positive for VTE increases, thus increasing its positive predictive value. However, this has not been
clinically validated. When the PTP score decreases, the probability that a negative D-dimer test is a true
negative for VTE increases, thus increasing the NPV.96
If the D-dimer assay is validated in clinical studies for use with a PTP in excluding VTE, the need for
further testing may be reduced with a potential for reduction in cost.99,100 The result of the D-dimer assay
is interpreted in relation to the established threshold for the method. The threshold concentration
established for one method is not transferable to another method. A negative result (below the established
threshold) coupled with a low or intermediate risk (unlikely) score is considered justification for ruling
out VTE. A positive result (above the established threshold) indicates the presence of an abnormally high
concentration of the degradation products of fibrin but is not specific for VTE. Further evaluation may be
needed (see the algorithm in Figure 1).
10.1 Result Reporting
Some manufacturers report D-dimer concentration in DDUs, which are based on the amount of D-dimer
fragment present, whereas others use FEUs. It is recommended that laboratories report the type and
magnitude of units recommended by the manufacturer, and do not perform any conversion of units. Use
of FEU equates the mass of a D-dimer fragment to the mass of the fibrinogen molecule from which it is
derived. (Thus, the mass of a D-dimer fragment expressed in FEUs is approximately 1.7 times101 the mass
of the same fragment expressed in DDUs. In many clinical settings, it is common practice to convert the
values from one to the other using a factor of 2. As emphasized below, such mathematical conversions of
the data are not acceptable.) To complicate matters further, manufacturers may express the result in a
variety of magnitudes (eg, picograms per milliliter, nanograms per milliliter, micrograms per liter,
milligrams per liter). Although several manufacturers use a threshold value of 500 μg/L FEU for the
exclusion of DVT, a recent meta-analysis reported a wide range of thresholds.70 There is a misconception
among some clinicians that there exists a universal threshold value of 500 μg/L FEU for all D-dimer
assays. Laboratories should report the type of units used and threshold for the exclusion of VTE. It is
important that each manufacturer publishes the reporting units and threshold value for a method in the
product labeling (insert sheet) of the reagent and that each laboratory reports the numerical D-dimer result
value, along with the reporting type and magnitude of units for its specific method and the validated
threshold value for exclusion of VTE, which is the information provided by the manufacturer.
Mathematically converting the reported data to different units is not acceptable because it is associated
with increased errors in reporting.102,103
The purpose of this document is to provide guidance on the use of the results of the D-dimer assay for the
exclusion of VTE. Results used in this diagnostic context should be interpreted in terms of a validated
threshold value that has been demonstrated to provide the required NPV and sensitivity. The report should
include the result and the threshold value in the units appropriate for the specific assay used. Advice
regarding interpretation of the result with the PTP may also be useful. However, most laboratories use
results from a D-dimer assay for many diagnostic applications in addition to exclusion of VTE. In this
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setting, in addition to the RI, other information regarding the use of the test along with the criteria for
exclusion of VTE would be appropriate. The exact text used may vary among clinical settings.
If the laboratory does not use a D-dimer assay for exclusion of VTE, the threshold established for
exclusion of VTE is no longer appropriate. In these cases, the reporting format should include the result,
the RI, and sufficient additional information for the interpretation of the results for those purposes. The
exact text employed may vary from site to site.
Figure 1 presents an algorithmic approach using clinical PTP, D-dimer, and imaging studies to evaluate
VTE. Other similar approaches have been proposed. Michiels and colleagues104 reviewed methods using
combined clinical, laboratory, and imaging studies to reduce the need for repeat compression
ultrasonography in the evaluation for DVT. The use of these standardized approaches makes it possible to
limit patient exposure to invasive procedures, improve efficiency in evaluation of patients, and potentially
reduce cost.104
Pretest Probability Algorithm Score
Unlikely; Low or Likely;

Intermediate Probability High Probability
D-dimer: Negative D-dimer: Positive

(< Threshold Value) ( Threshold Value)
Imaging
Techniques
(-) (+)
No DVT or PE DVT or PE
Evaluation for other etiologies

Treatment
causing symptoms.*
Figure 1. DVT/PE PTP Algorithm

* When studies are done for research or regulatory purposes, or in some clinical cases for which the results are not clear, follow-
up to confirm the negative diagnosis may be necessary.
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antibodies. J Clin Pathol. 1984;37(8):882-887.
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venous thromboembolism. Chest. 2003;124(3):1116-1119.
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Lee-Lewandrowski E, Nichols J, Van Cott E, et al. Implementation of a rapid whole blood D-dimer test in the emergency department of an
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Lee-Lewandrowski E, Van Cott EM. Evaluation of the Biosite Triage® quantitative whole blood D-dimer assay and comparison with the
bioMerieux VIDAS® D-dimer exclusion test: validation and utility for use in the central laboratory and at the point of care. Point of Care.
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of an academic medical center: impact on test utilization and ED length of stay. Am J Clin Pathol. 2008;129(5):796-801.
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Freyburger G, Reboul MP, Labrouche S, Saillour F, Grenier N. Diagnosis accuracy of a new challenger for thrombosis exclusion, the
Stratus CS DDMR. Clin Chim Acta. 2005;354(1-2):181-189.
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D-dimer) for the exclusion of venous thromboembolism. Blood Coagul Fibrinolysis. 2004;15(5):435-438.
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Wayne, PA: Clinical and Laboratory Standards Institute; 2005.
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document C28-A3c. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.
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Michiels JJ, Kasbergen H, Oudega R, et al. Exclusion and diagnosis of deep vein thrombosis in outpatients by sequential noninvasive tools.
Intl Angiol. 2002;21(1):9-19.
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Michiels JJ, Freyburger G, van der Graaf F, Janssen M, Oortwijn W, van Beek EJ. Strategies for the safe and effective exclusion and
diagnosis of deep vein thrombosis by the sequential use of clinical score, D-dimer testing, and compression ultrasonography. Semin Thromb
Hemost. 2000;26(6):657-667.
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Appendix. US Food and Drug Administration Clinical Study Design: An Example
A1. US Food and Drug Administration Levels of Clearance
When a D-dimer assay is cleared by the US Food and Drug Administration (FDA), the labeling for
“Indications for Use” may be either for “Aid in Diagnosis of Deep Vein/Venous Thrombosis (DVT) or
Pulmonary Embolism/Embolus (PE)” or for “Exclusion of DVT or PE.” These are not the same levels of
clearance; the differences in the clearance for these “Indications for Use” are listed in Table A1.
Table A1. Example of the Level of Supporting Evidence Used to Discriminate Between “Aid in
Diagnosis of VTE” vs “Exclusion of VTE” Product “Indications for Use” Claims
“Indications for Use” Product Claims
Discrimination Criteria Aid in Diagnosis of VTE Exclusion of VTE
Forming diagnosis Interpretation of the D-dimer Interpretation of the D-dimer result
result in clinical context in conjunction with pretest
probability (PTP)
External studies Outcome study Management study
Number of external study sites Minimum of three sites Minimum of three sites
Number of samples Statistically significant number Statistically significant number of
of outpatient samples with consecutively eligible outpatients
known venous presented to an emergency room
thromboembolism (VTE) (ER) or outpatient clinic with
diagnosis (>10% prevalence for suspicion of VTE (>10%
both DVT and PE) prevalence for both DVT and PE)
Validation criteria D-dimer values are compared to D-dimer values are compared to
a predicate D-dimer device final outcome (clinical truth)
through imaging techniques and, if
the imaging studies fail to confirm
VTE, the three-month patient
follow-up to confirm a negative
result.
Sensitivity Not defined >95%
Negative predictive value >97% >97%
(NPV)
Low end of 95% confidence >95% >95%
interval (CI) of the NPV
Low end of 95% CI of
Not defined >90%
sensitivity
A2. US Food and Drug Administration Clinical Study
The information below is intended to provide guidance to test developers on clinical study design of D-
dimer assays intended for use in the “aid in diagnosis” of or “exclusion” of DVT and PE.
A3. US Food and Drug Administration Patient Population
Clearly delineate and outline patient inclusion/exclusion criteria before the study is conducted. Select the
study population from prospective, consecutive ambulatory outpatients (generally presenting to an ER or
outpatient clinic) suspected of having VTE. Take care to reduce possible selection bias in the patient
population by selecting patients over a period of time.
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Appendix. (Continued)
Report demographic characteristics of the enrolled patients. Report the number of participants satisfying
the criteria for inclusion that did not undergo testing. Describe why participants failed to receive either
test.
A4. US Food and Drug Administration Sample Size
Base the sample size on the maximum sufficient number of samples to cover the range of the results (or at
least the clinically important range of the assay); capture biological variation in patients and important
patient subgroups; and provide appropriate power to meet the statistical end points in Table A1.
A5. US Food and Drug Administration Study Sites
Conduct the study at a minimum of three sites. Present study results by site and combine them if an
analysis of variance indicates the site data can be pooled.
A6. US Food and Drug Administration Clinical Assessment
Assess enrolled patients according to an established, predetermined clinical PTP model (eg, Wells,
Geneva). All testing sites should follow the same PTP model.
A7. US Food and Drug Administration Clinical Reference Testing
Clinical reference testing refers to objective imaging studies for DVT or PE. Ideally, diagnose all patients
following the same clinical algorithm at all clinical sites. If this is not possible, then each site should fully
disclose the algorithm used to evaluate for DVT or PE. If a D-dimer result is used as part of the site’s
algorithm to evaluate for DVT or PE, then the assay must be one previously cleared by the appropriate
regulatory agency for a DVT or PE exclusionary claim.
The proposed D-dimer assay should not be a part of the clinical assessment used to diagnose the patient.
A8. US Food and Drug Administration Follow-up
Follow patients testing D-dimer negative with a low or low/intermediate (DVT or PE unlikely) PTP for a
minimum of three months to monitor development of thrombosis before declaring a true negative.
Exclude results from patients without a finalized follow-up.
A9. US Food and Drug Administration Statistical Analysis
Present the data as calculated estimates of sensitivity, specificity, positive predictive value (PPV), and
NPV, along with 95% CIs of each. The PPV should exceed the prevalence of disease in the population.
Calculate and report failure rates (patients with negative D-dimer and a nonhigh PTP who later develop
thrombosis during the follow-up period).
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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 approach is based on the model presented in the
most current edition of CLSI document HS01—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 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:
Documents and Records Equipment Information Management Process Improvement

Organization Purchasing and Inventory Occurrence Management Customer Service
Personnel Process Control Assessments—External Facilities and Safety
and Internal
H59-A addresses the QSEs 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.
Assessments–
and Inventory
Improvement
Facilities and
Organization
Management
Management
External and
and Records
Information
Occurrence
Documents
Purchasing
Equipment
Personnel
Customer
Internal
Control
Process
Process
Service
Safety
X
C28
EP05
EP07 EP07
EP12
EP15
H21
I/LA30
M29
Adapted from CLSI document HS01—A Quality Management System Model for Health Care.
Path of Workflow
A path of workflow is the description of the necessary steps to deliver the particular product or service that the
organization or entity provides. For example, CLSI document GP26⎯Application of a Quality Management System
Model for Laboratory Services defines a clinical laboratory path of workflow, which consists of three sequential
processes: preexamination, examination, and postexamination. All clinical laboratories follow these processes to
deliver the laboratory’s services, namely quality laboratory information.
H59-A 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

receipt/processing
Sample collection
Results reporting
Sample transport
Results review
and follow-up
and archiving
Interpretation
Examination
Examination
management
ordering
Sample
Sample
X X X X X X
H21 H21
Adapted from CLSI document HS01—A Quality Management System Model for Health Care.
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Related CLSI Reference Materials∗

C28-A3c Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved
Guideline—Third Edition (2008). This document contains guidelines for determining reference values and
reference intervals for quantitative clinical laboratory tests.
EP05-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.
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.
EP12-A2 User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline—Second Edition
(2008). This document provides a consistent approach for protocol design and data analysis when evaluating
qualitative diagnostic tests. Guidance is provided for both precision and method-comparison studies.
EP15-A2 User Verification of Performance for Precision and Trueness; Approved Guideline—Second Edition
(2005). 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.
H21-A5 Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation
Assays and Molecular Hemostasis Assays; Approved Guideline—Fifth Edition (2008). This document
provides procedures for collecting, transporting, and storing blood; processing blood specimens; storing
plasma for coagulation testing; and general recommendations for performing the tests.
I/LA30-A Immunoassay Interference by Endogenous Antibodies; Approved Guideline (2008). This guideline
discusses the nature and causes of interfering antibodies, as well as their effects on immunoassays and
mechanisms by which interference occurs. Methods to identify and characterize the interferences are
addressed along with assessment of methods used to eliminate interference.
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.
∗
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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NOTES
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NOTES
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Active Membership
(As of 1 January 2011)
Sustaining Members MA Dept. of Public Health Laboratories Integrated BioBank Al Rahba Hospital (United Arab Emirates)
Malaria Research Training Center IntelligentMDx, Inc. Alameda County Medical Center (CA)
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H59-A Quantitative Ddimer For The Exclusion of VTE

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H59-A Quantitative Ddimer For The Exclusion of VTE

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H59-A

Quantitative D-dimer for the Exclusion of

Licensed to: Paul D\'Orazio, PhD Instrumentation Laboratory

Licensed to: Paul D\'Orazio, PhD Instrumentation Laboratory

John D. Olson, MD, PhD C. Alex McMahan, PhD

Licensed to: Paul D\'Orazio, PhD Instrumentation Laboratory

ii D\'Orazio, PhD Instrumentation Laboratory

Consensus Committee on Hematology

Document Development Committee on Quantitative D-dimer

John D. Olson, MD, PhD Kathleen Trumbull, MS, Lois M. Schmidt, DA

Philippe de Moerloose, MD Alicia Rico-Lazarowski, H(ASCP)CM

Chris Gardiner, FIBMS, MSc, PhD Miguel Sales

C. Alex McMahan, PhD Linda Stang, MLT

Licensed to: Paul D\'Orazio, PhD Instrumentation Laboratory iii

iv D\'Orazio, PhD Instrumentation Laboratory

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

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

The Quality Management System Approach ........................................................................................ 30

Related CLSI Reference Materials ....................................................................................................... 31

vi D\'Orazio, PhD Instrumentation Laboratory

Licensed to: Paul D\'Orazio, PhD Instrumentation Laboratory vii

viiiD\'Orazio, PhD Instrumentation Laboratory

Quantitative D-dimer for the Exclusion of Venous Thromboembolic Disease;

4.1 A Note on Terminology

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

anticoagulant – an agent, natural or pharmacological, that inhibits clotting of blood or plasma.

fluorescence energy transfer immunoassay – a type of sandwich fluorescence immunoassay of an

fluorescence resonance energy transfer – a type of fluorescent immunoassay, in which a donor

imprecision – dispersion of independent results of measurements obtained under specified conditions;

measurand – quantity intended to be measured (ISO/IEC Guide 99).9

measurement procedure – detailed description of a measurement according to one or more measurement

nonlabeled immunoassays//microparticle agglutination immunoassay – a type of assay that is based

precision (measurement) – closeness of agreement between indications or measured quantity values

prevalence – the frequency of a condition of interest expressed as a percentage (number fraction

reproducibility (measurement) – measurement precision (closeness of agreement between indications or

4.3 Abbreviations and Acronyms

5 Overview of D-dimer Testing

5.1 Venous Thromboembolism Background

5.2 D-dimer Measurement

5.3 Problems With D-dimer Measurement

5.4 Harmonization vs Standardization

6 Patient Evaluation and Pretest Probability

Table 1. Wells Simplified Clinical Model for Assessment of DVT*,34

Table 2. Predicting Probability of PE: Comparison of Geneva and Wells Scores*,†,37

Clinical Probability Clinical Probability

6.1 Limitations Related to Patient Conditions or Therapies

• Fibrinolytic therapy within the previous seven days

7 Specimen Collection and Processing

7.1 Patient Variables/Preparation Before Collection

7.2 Specimen Collection

7.3 Specimen Processing, Transport, and Storage

Lipemic or hemolyzed samples may cause interference, especially in quantitative, microparticle

8.1 Quantitative Sandwich Assays

8.2 Quantitative Microparticle Agglutination

8.3 Semiquantitative Microparticle Agglutination Methods

8.4 Point-of-Care Testing

8.5 Other Technologies

9 Assay Characteristics, Reference Interval, and Threshold for Exclusion of

9.1 Recommended Assay Characteristics

9.2 Reference Interval

9.3 Establishing the Threshold for Venous Thromboembolism Exclusion: Comparison to

9.5 Satisfying Regulatory Requirements

10.1 Result Reporting