H60A - Laboratory Testing For Lupus Anticoagulant Testing Approved April 2014

April 2014
H60-A
Laboratory Testing for the Lupus
Anticoagulant; Approved Guideline
This document provides guidance and recommendations regarding

the proper collection and handling of the specimen; descriptions and
limitations of screening and confirmatory assays, and mixing tests
used to identify lupus anticoagulant (LA); determination of cutoff
values and calculations associated with the various assays; and
interpretation of test results in an LA panel.
A guideline for global application developed through the Clinical and Laboratory Standards Institute consensus process.
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ISBN 1-56238-959-9 (Print)
ISBN 1-56238-960-2 (Electronic)
ISSN 1558-6502 (Print) H60-A
ISSN 2162-2914 (Electronic) Vol. 34 No. 6
Laboratory Testing for the Lupus Anticoagulant; Approved Guideline
Volume 34 Number 6
Marlies Ledford-Kraemer, MBA, BS, MT(ASCP)SH Sandra C. Hollensead, MD
Gary W. Moore, BSc, DBMS, CSci, FIBMS, CBiol, MSB, Piet Meijer, PhD
CertMHS Karen A. Moffat, BEd, ART, FCSMLS(D)
Ralph Bottenus, PhD William L. Nichols, MD
John T. Brandt, MD Thomas L. Ortel, MD, PhD
Donna D. Castellone, MS, MT(ASCP)SH Michael J. Sanfelippo, MS, MT(ASCP)
Christine Daniele, MT(ASCP) Rosemary Grillo Scott
Philip G. de Groot, PhD Rita Selby, MBBS, FRCPC, MSc
François Depasse, PharmD, MSc Linda Stang, MLT
Jeffrey S. Dlott, MD, FCAP, FASCP Perumal Thiagarajan, MD
Thomas Exner, PhD Mark Triscott, PhD
Emmanuel J. Favaloro, PhD, FFSc (RCPA) Elizabeth M. Van Cott, MD
Robert C. Gosselin, CLS
Abstract
Identification of the lupus anticoagulant (LA) by laboratory testing is critical for diagnosing the antiphospholipid syndrome and
investigating unexpectedly prolonged activated partial thromboplastin time values. The “anticoagulant” effect of LA is restricted
to the prolongation of clotting times when using in vitro, clot-based coagulation assays that are used as surrogates for identifying
LA. Clinical and Laboratory Standards Institute document H60—Laboratory Testing for the Lupus Anticoagulant; Approved
Guideline provides guidance and recommendations regarding the proper collection and handling of the specimen; descriptions
and limitations of screening and confirmatory assays, and mixing tests used to identify LA; determination of cutoff values and
calculations associated with the various assays; and interpretation of test results in an LA panel. The guideline is provided for use
by laboratorians, physician stakeholders, manufacturers of LA assays, researchers, external quality assessment programs, and
accrediting and regulatory agencies. The intent of this guideline is to present information in a practical and easily understandable
format; thereby facilitating a standardized approach to LA testing, gaining acceptance in practice, and improving testing quality.
Clinical and Laboratory Standards Institute (CLSI). Laboratory Testing for the Lupus Anticoagulant; Approved Guideline. CLSI
document H60-A (ISBN 1-56238-959-9 [Print]; ISBN 1-56238-960-2 [Electronic]). Clinical and Laboratory Standards Institute,
950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087 USA, 2014.
The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through
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Number 6 H60-A
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Suggested Citation
CLSI. Laboratory Testing for the Lupus Anticoagulant; Approved Guideline. CLSI document H60-A.
Wayne, PA: Clinical and Laboratory Standards Institute; 2014.
Approved Guideline
April 2014
ISBN 1-56238-959-9 (Print)

ISBN 1-56238-960-2 (Electronic)
ISSN 1558-6502 (Print)
ISSN 2162-2914 (Electronic)
ii
Volume 34 H60-A
Committee Membership
Consensus Committee on Hematology

Richard A. Marlar, PhD Ian A. Giles, MD Hans J. Hoffmann, PhD
Chairholder Sysmex America, Inc. Abbott GmbH
Oklahoma City VA Medical Mundelein, Illinois, USA Wiesbaden-Delkenheim, Germany
Center
Oklahoma City, Oklahoma, USA Robert C. Gosselin, CLS Loveleen Chatha Kang, MD
University of California, Davis College of American Pathologists
Mike Keeney, ART, FCSMLS(D) Health Northfield, Illinois, USA
Vice-Chairholder System
London Health Sciences Center Sacramento, California, USA
London, Ontario, Canada
Kathleen M. Curran, BSMT

New York State Department of
Health
Albany, New York, USA
Document Development Committee on Laboratory Testing for the Lupus Anticoagulant
Marlies Ledford-Kraemer, MBA, Philip G. de Groot, PhD Staff

BS, MT(ASCP)SH University Medical Center
Chairholder Utrecht, The Netherlands Clinical and Laboratory Standards
CLOT-ED, Inc. Institute
Islamorada, Florida, USA Thomas Exner, PhD Wayne, Pennsylvania, USA
Haematex Research Pty Ltd
Gary W. Moore, BSc, DBMS, Sydney, Australia Luann Ochs, MS
CSci, FIBMS, CBiol, MSB, Senior Vice President – Operations
CertMHS Emmanuel J. Favaloro, PhD, FFSc
Vice-Chairholder (RCPA) Jennifer K. Adams, MT(ASCP),
GSTS Pathology ICPMR, Westmead Hospital MSHA
London, United Kingdom New South Wales, Australia Staff Liaison
Ralph Bottenus, PhD Karen A. Moffat, BEd, ART, David E. Sterry, MT(ASCP)
Instrumentation Laboratory FCSMLS(D) Project Manager
Orangeburg, New York, USA Hamilton Regional Laboratory
Medicine Program Megan L. Tertel, MA
Christine Daniele, MT(ASCP) West Hamilton, Ontario, Canada Editor
Diagnostica Stago, Inc.
Parsippany, New Jersey, USA William L. Nichols, MD Joanne P. Christopher, MA
Mayo Clinic Assistant Editor
Rochester, Minnesota, USA
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Acknowledgment
CLSI and the Consensus Committee on Hematology gratefully acknowledge the following individuals for
their contributions during the development of this document:
John T. Brandt, MD Sandra C. Hollensead, MD Rita Selby, MBBS, FRCPC,

Wenatchee, Washington, University of Louisville MSc
USA School of Medicine Sunnybrook Health Sciences
Louisville, Kentucky, USA Centre & University Health
Donna D. Castellone, MS, Network
MT(ASCP)SH Piet Meijer, PhD Toronto, Ontario, Canada
Roche Diagnostics ECAT Foundation
Indianapolis, Indiana, USA Leiden, The Netherlands Linda Stang, MLT
University of Alberta
François Depasse, PharmD, Thomas L. Ortel, MD, PhD Hospital
MSc Duke University Medical Edmonton, Alberta, Canada
Diagnostica Stago Center
Asnieres Sur Seine, France Durham, North Carolina, Perumal Thiagarajan, MD
USA Michael E. DeBakey VA
Jeffrey S. Dlott, MD, FCAP, Medical Center
FASCP Michael J. Sanfelippo, MS, Houston, Texas, USA
Quest Diagnostics, Inc. MT(ASCP)
Chantilly, Virginia, USA Marshfield Labs Mark Triscott, PhD
Marshfield, Wisconsin, USA Instrumentation Laboratory
Robert C. Gosselin, CLS Orangeburg, New York, USA
University of California, Rosemary Grillo Scott
Davis Health System Diagnostica Stago Elizabeth M. Van Cott, MD
Sacramento, California, USA Parsippany, New Jersey, Massachusetts General
USA Hospital
Boston, Massachusetts, USA
The authors would like to dedicate this document to the memory of Douglas A. Triplett, MD, whose work
in lupus anticoagulant testing and the antiphospholipid syndrome has left an indelible mark. His
innumerable contributions through scientific research and education have guided many in this field. Dr.
Triplett’s name is synonymous with lupus anticoagulant testing and we are indebted to him for his efforts
and commitment to this field.
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Contents
Abstract ....................................................................................................................................................i
Committee Membership........................................................................................................................ iii
Foreword ................................................................................................................................................ix
1 Scope.......................................................................................................................................... 1
2 Introduction ................................................................................................................................ 1
2.1 Historical Perspective of Antiphospholipid Syndrome and Relationship to Lupus
Anticoagulant Testing ................................................................................................................ 3
3 Standard Precautions .................................................................................................................. 4
4 Terminology............................................................................................................................... 4
4.1 A Note on Terminology ................................................................................................ 4
4.2 Definitions .................................................................................................................... 5
4.3 Abbreviations and Acronyms ....................................................................................... 8
5 Equipment .................................................................................................................................. 9
6 Validation of Assay Systems ................................................................................................... 10

6.1 Imprecision ................................................................................................................. 10
6.2 Inaccuracy ................................................................................................................... 11
6.3 Establishment of Reference Intervals and Cutoff Values ........................................... 12
6.4 Analytical Specificity ................................................................................................. 13
6.5 Diagnostic Sensitivity and Diagnostic Specificity ...................................................... 14
7 Preexamination Issues (Criterion A) ........................................................................................ 14
7.1 Patient Selection ......................................................................................................... 15
7.2 Specimen Collection and Transport ............................................................................ 17
7.3 Sample Preparation ..................................................................................................... 18
8 Preliminary Examination Issues .............................................................................................. 21
8.1 APTT .......................................................................................................................... 21
8.2 Prothrombin Time-International Normalized Ratio.................................................... 23
8.3 Thrombin Time ........................................................................................................... 24
9 Principles of Lupus Anticoagulant Assays .............................................................................. 25
9.1 Intrinsic Pathway Assays (see Appendix C) ............................................................... 25
9.2 Common Pathway Assays (see Appendix C) ............................................................. 27
9.3 Extrinsic Pathway Assays (see Appendix C) .............................................................. 28
9.4 Overview of Assay Performance ................................................................................ 29
10 Assays to Screen for the Presence of Lupus Anticoagulant (Criterion B) ............................... 31
10.1 Available Screening Assays and Their Usage ............................................................ 31
10.2 APTT .......................................................................................................................... 32
10.3 APTT-based Silica Clotting Time .............................................................................. 33
10.4 Dilute Russell’s Viper Venom Time........................................................................... 34
10.5 Dilute Prothrombin Time ............................................................................................ 35
10.6 Kaolin Clotting Time .................................................................................................. 36
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Contents (Continued)
11 Assays to Confirm the Presence of Lupus Anticoagulant (Criterion C) .................................. 37
11.1 APTT-based Platelet Neutralization Procedure .......................................................... 38
11.2 APTT-based Hexagonal Phase Phospholipid Neutralization Test .............................. 38
11.3 APTT-based Silica Clotting Time Confirmatory Test ................................................ 39
11.4 Dilute Russell’s Viper Venom Time Confirmatory Test ............................................ 40
11.5 Dilute Prothrombin Time Confirmatory Test ............................................................. 41
12 Mixing Test as Applied to Screening, Confirmatory, and Integrated Assays (Criterion D) .... 42
12.1 Reasons for Performing a Mixing Test ....................................................................... 43
12.2 Normal Pooled Plasma ................................................................................................ 44
12.3 Calculation of Test Results ......................................................................................... 45
12.4 Limitations of Mixing Test ......................................................................................... 46
13 Postexamination Issues ............................................................................................................ 47
13.1 Interpretation of Preliminary Examination Assays ..................................................... 48
13.2 Interpretation of Lupus Anticoagulant Screening Assays
(Criterion/Recommendation B) .................................................................................... 49
13.3 Interpretation of Lupus Anticoagulant Confirmatory Assays
(Criterion/Recommendation C) .................................................................................. 50
13.4 Interpretation of Lupus Anticoagulant Mixing Tests
(Criterion/Recommendation D) .................................................................................. 52
13.5 Interpretation of Lupus Anticoagulant Panel (Criterion/Recommendation F) ........... 53
13.6 How to Report Test Results (Criterion/Recommendation F)...................................... 54
14 Quality Control and Quality Assurance ................................................................................... 54
14.1 Reagent Lot-to-Lot Number Verification ................................................................... 54
14.2 Internal Quality Control .............................................................................................. 55
14.3 External Quality Assessment ...................................................................................... 55
15 Harmonization of CLSI Lupus Anticoagulant Guideline With Other Guidelines ................... 56
References ............................................................................................................................................. 58
Appendix A. Algorithmic Approach to Lupus Anticoagulant Testing ................................................. 71
Appendix B. Establishing Reference Intervals and Cutoff Values ....................................................... 74
Appendix C. Lupus Anticoagulant Testing in Relationship to Coagulation Pathways ........................ 75
Appendix D. Diagrammatic Principles for Lupus Anticoagulant Tests................................................ 76
Appendix E. Table of Interferences and Limitations for Specific Lupus Anticoagulant Tests ............ 84
Appendix F. Formulas and Examples for Calculating Test Results ..................................................... 88
Appendix G1. Interpretive Comments and Rationale for Comments Based on Patient Examples
(Data) ................................................................................................................................................... 89
Appendix G2. Interpretive Comments and Rationale for Comments Based on Patient Examples
(Comments)........................................................................................................................................... 90
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Contents (Continued)
The Quality Management System Approach ........................................................................................ 92
Related CLSI Reference Materials ....................................................................................................... 94
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Volume 34 H60-A
Foreword
Synopsis of Diagnostic Criteria and Testing Recommendations
Criteria for the Laboratory Diagnosis of the Lupus Anticoagulant
In order to make a laboratory diagnosis of the lupus anticoagulant, a sample should identify with the
following:
A. Procurement: adherence to standardized protocols for collection and processing of blood to be used
for testing
B. Screening: prolongation of at least one of two different phospholipid-dependent clotting assays based
on different principles and coagulation pathways
C. Confirmation: evidence that prolongation of the screening test(s) demonstrates phospholipid

dependence by using a similar second test(s) using altered concentrations and/or composition of
phospholipids
D. Mixing: if mixing assays are performed, evidence of inhibitory activity shown by the effect of patient
plasma on an equal volume of normal pooled plasma
E. Exclusion: distinguishing the lupus anticoagulant from other causes of prolonged clotting times that
may mask, mimic, or coexist with the lupus anticoagulant, such as anticoagulant therapies or other
coagulopathies
F. Interpretation and Reporting: numerical results of all testing should be reported, and interpretive
comments that address and integrate these results should be provided
Recommendations Specific to Each Criterion for the Laboratory Diagnosis of the Lupus
Anticoagulant
A. Procurement
1. Testing should preferably be performed in the absence of anticoagulant therapy (except for
antiplatelet therapy).
2. Ideally, samples should not be obtained from vascular access devices.
3. Platelet count of patient-citrated platelet-poor plasma should be < 10 × 109/L.
4. Testing may be performed on fresh or properly frozen/thawed samples.
B. Screening Assays
1. Two tests, representing different principles and coagulation pathways, that are known to be
responsive to the lupus anticoagulant (eg, low phospholipid concentrations) should be used to
screen for the lupus anticoagulant.
2. Lupus anticoagulant–responsive activated partial thromboplastin time and dilute Russell’s viper
venom time tests are recommended as the preferred minimal screening assays.
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Number 6 H60-A
3. Other tests for the lupus anticoagulant referenced in this document may supplement the preferred
minimal screening tests.
4. Where test design permits, results should be calculated using the mean of the reference interval
and reported as a normalized ratio.
5. Routine coagulation tests, prothrombin time-international normalized ratio, activated partial

thromboplastin time, and thrombin time, as indicated, may help to characterize anticoagulant
effects (eg, heparin, vitamin K antagonists, direct thrombin inhibitors, factor Xa inhibitors) or
sample suitability (eg, serum sample, improper anticoagulant tube) for lupus anticoagulant
testing and interpretation.
C. Confirmatory Assays
1. Confirmatory assays should use the same assay principle as the screening test that was initially
found to be abnormal (eg, if the dilute Russell’s viper venom time test is abnormal, then a dilute
Russell’s viper venom time test–based confirmatory assay should be used).
2. For paired tests, results should be calculated using the mean of the reference interval for each
screening and confirmatory test and reported as a normalized screen to confirm ratio or
indication of percentage correction of screen ratio by confirm ratio.
3. Solid-phase immunoassays for antibodies against phospholipid (eg, anti-cardiolipin or anti-β2

glycoprotein I) should not be considered as lupus anticoagulant confirmatory procedures.
D. Mixing Test (if performed)
1. The platelet count of the normal pooled plasma should be < 10 × 109/L.
2. A mix ratio of one part plasma sample to one part normal pooled plasma is recommended as the
preferred ratio for a mixing test.
3. The dilution effect of a 1:1 mixing test may mask lupus anticoagulant inhibitory activity. Other
mix ratios (eg, four parts plasma sample to one part normal pooled plasma) can be used, if
validated by the laboratory.
4. Mixing test inhibition is assessed by either comparison of normalized ratios to cutoff values
specific for each lupus anticoagulant screening or confirmatory mixing test or by calculating an
index of circulating anticoagulant.
5. Incubated mixing tests are not recommended for routine lupus anticoagulant testing, but should
be performed when indicated (eg, when a specific factor inhibitor is suspected).
E. Exclusion
1. The lupus anticoagulant should be distinguished from anticoagulant therapies and/or other
coagulation disorders that may interfere with lupus anticoagulant testing and interpretation.
2. If possible, perform factor assays whenever there is suspicion of a specific factor deficiency or
inhibitor, using three or more dilutions of patient plasma and an activated partial thromboplastin
time reagent that is unresponsive to the lupus anticoagulant.
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Volume 34 H60-A
F. Interpretation and Reporting
1. Numerical results of all testing should be reported with the reference interval or cutoff value.
2. Interpretive comments that address and integrate all test results (the lupus anticoagulant panel)
should be provided.
3. The interpretive report should indicate whether the lupus anticoagulant is present, not detected,
or indeterminate.
4. Solid-phase assays for antibodies against cardiolipin and/or anti-β2 glycoprotein I are
recommended as part of an evaluation for antiphospholipid syndrome.
5. If the lupus anticoagulant is present, the test panel should be repeated at or beyond 12 weeks to
determine persistence of the lupus anticoagulant as part of the evaluation for antiphospholipid
syndrome.
Key Words
Antiphospholipid syndrome, lupus anticoagulant, lupus anticoagulant confirmatory assays, lupus

anticoagulant screening assays, mixing test
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Volume 34 H60-A
Laboratory Testing for the Lupus Anticoagulant; Approved Guideline
1 Scope
This document provides guidance for the performance and interpretation of screening assays,
confirmatory assays, and mixing tests used to identify the lupus anticoagulant (LA). It is intended to assist
in standardization of LA testing and it addresses preexamination issues, examination concerns, and
postexamination matters that pertain to interpretation of individual tests or combinations of assays.
Recommendations from this guideline, when feasible, harmonize with other national and international
guidelines currently in existence. Taken together, standardization and harmonization will permit
laboratories to improve the quality and interpretation of their LA testing.
The intended users of this guideline are laboratory personnel responsible for performing LA testing,
physicians (eg, hematologists, pathologists, rheumatologists), external quality assessment (EQA)
programs, researchers, and manufacturers of reagents used in LA testing.
Two types of methodologies are used for the diagnosis of the antiphospholipid syndrome (APS). This
guideline is limited to clot-based coagulation assays used as surrogates for identifying LA—a strong risk
factor for thrombosis. The guideline will not address solid-phase testing for anti-phospholipid (aPL) (eg,
anti-cardiolipin [aCL] or anti-β2 glycoprotein I [aβ2GPI]), because detection of these specific antibodies
may or may not relate to the laboratory anomaly of a prolonged activated partial thromboplastin time
(APTT).
2 Introduction
Identification of LA by laboratory testing is critical for investigating unexpectedly prolonged APTT
values and diagnosing APS. According to recent consensus classification criteria, two conditions must be
met for defining APS: 1) the persistent presence of circulating aPL in plasma and/or serum, and 2) a
history of thrombosis and/or pregnancy morbidity including fetal loss.1,2 APS is an autoimmune disorder
that occurs in patients who, in general, also show laboratory evidence of antibodies directed against
plasma proteins that have an affinity for anionic phospholipids (PL). The dominant antigenic targets
recognized by aPL in patients with APS are β2GPI or prothrombin.3-5
The terms APS and LA are both misnomers and LA itself is a double misnomer.6,7 The autoantibodies
associated with APS are not directed against PL in general but specifically against proteins that bind to
anionic PL. LA comprises a heterogeneous group of autoantibodies that can develop in individuals with
autoimmune conditions (systemic lupus erythematosus [SLE], APS, or other autoimmune disorders) or
can arise spontaneously.8-10 LA can also be found transiently in plasma from patients with infections or
malignancies, or from patients using certain drugs. The “anticoagulant” effect of LA is restricted to the
prolongation of clotting times (competition between these antibodies and coagulation proteins for PL
surfaces) in clot-based assays (ie, in vitro), whereas in vivo, these antibodies are variably associated with
thrombosis.11,12 Persistent LA is the most important acquired risk factor for thrombosis (or its recurrence)
in APS.8,13 LA may also cause bleeding due to immune-mediated deficiencies of coagulation factors II
(FII) or X (FX) (see Section 7.1.1). Infection- or drug-induced LA tend to be transient, disappearing after
the infection resolves or when the medication is discontinued. Transient LA due to infections have rarely
been reported to increase thrombotic risk in association with immune-mediated protein S deficiency (see
Section 7.1.1). LA due to certain medications may not be transient and can increase thrombotic risk.14 LA
associated with malignancy might also resolve after the malignancy is treated.15,16
©
Clinical and Laboratory Standards Institute. All rights reserved. 1
Number 6 H60-A
Two types of laboratory testing are used in making a diagnosis of APS:
 Solid-phase assays for aCL and aβ2GPI

 Liquid-phase (plasma-based) assays for LA
Solid-phase (commonly ELISA) methods are used to identify and quantify the antibody isotypes
(immunoglobulin G [IgG], immunoglobulin M [IgM], immunoglobulin A [IgA]) to β2GPI, cardiolipin
(either dependent or independent of β2GPI), and prothrombin. These assays will not be discussed in this
document.
This guideline will focus on the detection of LA activity (antibodies against β2GPI/PL and/or antibodies
against prothrombin/PL) by surrogate, functional, liquid-phase, clot-based assays. These tests are
associated with all three laboratory pathways of coagulation: intrinsic, common, and extrinsic. Numerous
assays have been designed through the years, as shown in Table 1. Though potentially promising, many
have not been widely used or commercialized.17-20 Commercially available assays, in general, manipulate
PL concentration (or type) that permits LA to either showcase inhibitory action or to have this activity
neutralized. Hence, test systems have been segregated into those having “low” levels of PL to screen or
“tease out” the inhibitory action of LA vs those assays in which high concentrations of PL neutralize the
inhibitory effect. Due to antibody heterogeneity, different clot-based assays can detect different antibody
populations in the same patient.21,22 LA is considered to be more strongly associated with thrombotic risk
than ELISA assays for aCL and aβ2GPI.8,23 A provocative concept is that this stronger association may be
related to antibody titers, ie, a relatively high concentration of antibodies is required to prolong the
clotting time of LA assays, which are analytically relatively insensitive.24
Consensus criteria for the classification (not diagnosis) of APS were initially outlined in 1999 1 and
updated in 2006.2 Both documents include laboratory criteria for LA as set forth by the Scientific and
Standardization Committee (SSC) on Lupus Anticoagulant/Phospholipid-Dependent Antibodies of the
International Society on Thrombosis and Haemostasis (ISTH). Guidelines for LA testing from the ISTH
were first published in 1983,25 updated in 199126 and 1995,27 and revised in 2009.28 Guidelines are also
available globally from various national and international organizations.9,29,30 Many guidelines have built
upon recommendations and criteria from preceding works. This guideline seeks to continue this building
process while harmonizing with, and adding clarity to, the current guidelines. This will aid laboratories in
the laboratory diagnosis of LA and to assist clinicians in determining the presence of APS.
This guideline also introduces new paradigms for LA testing. The key shift is in the order in which tests
are performed: screening, confirmation, and mixing. This guideline recognizes that many interferences
affect the mixing test as shown in recent publications addressing the potential for false-negative or false-
positive test results when significant reliance is placed on the mixing test. Therefore, this guideline
suggests that less importance be assigned to the mixing test and places it last in the sequence of testing.
Additionally, this guideline stresses the need for and understanding of test principles and their association
with the three coagulation pathways. Thus, screening assays should not only be performed using two test
principles, but each should represent a different coagulation pathway, as recommended by the document
development committee. Finally, this guideline acknowledges the importance of not only performing an
LA panel of tests, but interpreting LA tests in total rather than in isolation. The need for appropriate cutoff
values that differentiate whether LA is or is not detected is inherent in the interpretation of LA tests.
Numerous sections of this guideline strive to reinforce this point.
©
2 Clinical and Laboratory Standards Institute. All rights reserved.
Volume 34 H60-A
Table 1. Historical Perspective of Laboratory Tests Used for the Detection of LA

Test Year LA Test Type Plasma Type Reference
PT 1935 NA Neat 155
PTT 1953 NA Neat 146
KCT 1958 NA Neat 147
APTT 1961 Independent Screening Neat 186
TTI 1976 Paired Neat 181
KCT 1978 Independent Screening Diluted 187
PNP 1983 Independent Confirmatory Neat 190
dAPTT 1985 Independent Screening Neat 183
dRVVT 1986 Paired Neat 207
SCT 1992 Paired Neat 185
HPNT 1993 Integrated Diluted 197
Textarin/Ecarin 1993 Paired Neat 211
APTT lupus ratio test 1993 Integrated Diluted 204
TSVT 1994 Independent Screening Neat 213
dPT 1994 Paired Neat 91
ASLA 2002 Paired Neat 234
dPT lupus ratio test 2002 Integrated Diluted 221
Abbreviations: APTT, activated partial thromboplastin time; ASLA, activated seven lupus anticoagulant; dAPTT, dilute activated
partial thromboplastin time; dPT, dilute prothrombin time; dRVVT, dilute Russell’s viper venom time; HPNT, hexagonal phase
phospholipid neutralization test; KCT, kaolin clotting time; LA, lupus anticoagulant(s); NA, not applicable; PNP, platelet
neutralization procedure; PT, prothrombin time; PTT, partial thromboplastin time; SCT, silica clotting time; TTI, tissue
thromboplastin inhibition; TSVT, Taipan snake venom time.
2.1 Historical Perspective of Antiphospholipid Syndrome and Relationship to Lupus

Anticoagulant Testing
APS is an example of a syndrome defined first by laboratory features (clotting assays, serology) and
subsequently characterized clinically.31 In 1952, Moore and Mohr observed that biologic false-positive
serological tests for syphilis (BFP-STS) occurred when using either a modified Wasserman or VDRL
test.32 The antigen used by both assays was a nontreponemal material, termed “lipoidal,” from the alcohol
extracts of beef heart tissue.33,34 Cardiolipin (1,3-bis(sn-3'-phosphatidyl)-sn-glycerol), an acidic
(negatively charged) PL and lecithin were purified from this “lipoidal” material.35 A second observation
made in 1952 was made by Conley and Hartmann who reported on two patients with SLE and BFP-STS
who had a hemorrhagic disorder.36 This disorder was attributed to a circulating anticoagulant that
markedly prolonged whole blood clotting times by “retarding the conversion of prothrombin to
thrombin.” In 1957, Laurell and Nilsson made the link between BFP-STS and a circulating
anticoagulant.37 Work by Bowie and colleagues in 1963 recognized the paradoxical association of a
circulating anticoagulant with thrombosis in SLE patients.38 It was not until 1972 that Feinstein and
Rapaport coined the term “lupus anticoagulant” based on its prevalence in SLE patients.39
In 1983, Harris and colleagues described a radioimmunoassay (by 1985 it had been converted into an
ELISA, the antecedent of those in use today) for antibodies against cardiolipin that was 200 to 400 times
more sensitive than the VDRL test. They noted that more than 60% of samples from SLE patients had
high levels of aCL. Moreover, there was a strong correlation between elevated aCL levels and 1) the LA,
2) venous and arterial thrombosis, and 3) thrombocytopenia. The perception was that the specificity of
aPL was for negatively charged PL in general rather than for cardiolipin in particular.40 Shortly thereafter,
two groups independently showed that aCL and LA comprised separate antibody groups.41,42
In 1985, Hughes43,44 suggested the term “anticardiolipin syndrome: to describe the association between
aCL and features such as venous/arterial thrombosis, recurrent fetal loss, neurological disease, pulmonary
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hypertension, and livedo reticularis.” Though sometimes referred to as the Hughes syndrome, APS was
later coined to emphasize that a group of aPL, not only aCL, was present in these patients.45,46
In 1990, three groups3,47,48 identified β2GPI, also termed apolipoprotein H, as a critical plasma protein
required for the binding of aCL to cardiolipin. This “cofactor” was necessary because cardiolipin, due to
its small molecular size (a hapten), could not by itself be immunogenic. These studies demonstrated that
purified aCL from patients with APS could only bind to cardiolipin in the presence of β2GPI (β2GPI-
dependent aCL). In contrast, aCL found in syphilis bound to cardiolipin in the absence of β2GPI (β2GPI-
independent aCL). Therefore, aCL associated with autoimmune disease (and increased risk for
thrombosis) could be distinguished from aCL found in syphilis and other infectious diseases such as
tuberculosis, hepatitis, and AIDS that generally are not associated with a thrombotic risk. In 1991, Bevers
and colleagues noted that LA are not directed to PL alone, but to prothrombin subsequent to its
Ca+2-mediated binding to PL.4 These findings align with observations that LA/aPL have a proclivity for
numerous serine proteases associated with enzyme-cofactor-substrate complexes assembled on PL
membranes.49-53
The in vivo mechanisms responsible for thrombosis and fetal loss in patients with APS are unknown,
although several potential theories have been suggested. Patients with LA activity that is β2GPI-
dependent are at high risk for developing thrombosis.13 The binding of β2GPI to anionic PL leads to a
conformational change in the protein that exposes new epitopes to which autoantibodies can bind.54,55
These autoantibodies confer a gain of function property to β2GPI by increasing its affinity for anionic PL.
This may help explain the role of β2GPI in the pathophysiology of APS. For example, anionic PL are
prevalent in membranes of many cells such as endothelial cells, monocytes, and platelets. Once activated,
these cells release products that downstream can lead to a hypercoagulable state. In vitro, the affinity of
aβ2GPI-β2GPI complexes for anionic PL is of a magnitude that can directly compete with coagulation
clotting factors for the catalytic PL required for clot-based assays, thereby prolonging clotting times.56
3 Standard Precautions
Because it is often impossible to know what isolates or specimens might be infectious, all patient and
laboratory specimens are treated as infectious and handled according to “standard precautions.” Standard
precautions are guidelines that combine the major features of “universal precautions and body substance
isolation” practices. Standard precautions cover the transmission of all known infectious agents and thus
are more comprehensive than universal precautions, which are intended to apply only to transmission of
blood-borne pathogens. The Centers for Disease Control and Prevention address this topic in published
guidelines that address the daily operations of diagnostic medicine in human and animal medicine while
encouraging a culture of safety in the laboratory.57 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 M29.58
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
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guidelines focuses on harmonization of terms to facilitate the global application of standards and
guidelines.
Verification focuses on whether specifications of a measurement procedure can be achieved, whereas

validation verifies that the procedure is fit for purpose.
In describing antibodies directed against (to) phospholipids, in general, or to a specific phospholipid, this
document uses the commonly accepted shortcut “anti.” Therefore, anti-β2GPI, anti-cardiolipin, and anti-
phospholipid are shortcuts for “antibodies against β2 glycoprotein I,” “antibodies against cardiolipin,”
and “antibodies against phospholipid,” respectively. In abbreviated form, the prefix “anti” is represented
by “a” (for example, aCL is the abbreviation for anti-cardiolipin).
4.2 Definitions
accuracy (measurement) – closeness of agreement between a measured quantity value and a true
quantity value of a measurand (JCGM 200:2012).59
acute phase reaction – the physiological response to inflammation, injury, illness, or stress, in which a
variety of plasma proteins become increased in concentration, or, in the case of some proteins, decreased
in concentration; NOTE: A subset of these proteins affects coagulation, eg, factor VIII, fibrinogen, and
von Willebrand factor become elevated.
analyte – component represented in the name of a measurable quantity (ISO 17511).60
analytical specificity – ability of a measurement procedure to measure solely the measurand (ISO
17511)60; NOTE: Specificity has no numerical value in this context.
anticoagulant – a substance that prevents coagulation, ie, it inhibits blood or plasma from clotting.
bias – the difference between the expectation of the test results and an accepted reference value (ISO
5725-1).61
cutoff value – the quantitative value of an analyte (eg, the upper limit of a reference interval for a
particular analyte) that is used to decide whether the result is above or below a clinical or analytical
decision point (ie, usually positive or negative, but may also represent divisions between grades of
positivity [eg, weak positive, moderate positive, strong positive]).
diagnostic sensitivity – the proportion of patients with a well-defined clinical disorder (or condition of
interest) whose test values are positive or exceed a defined decision limit (ie, a positive result and
identification of the patients who have a disease); NOTE 1: The clinical disorder must be defined by
criteria independent of the test under consideration; NOTE 2: The term “diagnostic sensitivity” (Europe)
is equivalent to “clinical sensitivity” (United States).
diagnostic specificity – the proportion of subjects who do not have a specified clinical disorder (or
condition of interest) whose test results are negative or within the defined decision limit; NOTE 1: This
term is equivalent to the US term “clinical specificity”; NOTE 2: In laboratory testing, the ability of a test
to give a negative result for patients who do not have the disease or condition for which they are being
tested. It is measured as the ratio of negative tests to the total number of tests in those who do not have the
disease or condition, and expressed as a percentage.
error (measurement)//measurement error – measured quantity value minus a reference quantity value
(JCGM 200:2012).59
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false-negative result – negative test result for a subject in whom the disease or condition of interest is
present; NOTE 1: Alternatively, a person who has a condition but is incorrectly identified as negative for
having the condition; NOTE 2: The term is often unclear in lupus anticoagulant testing because a gold
standard test is lacking.
false-positive result – positive test result for a subject in whom the disease or condition of interest is
absent; NOTE 1: Alternatively, a person who does not have the condition but is incorrectly identified as
positive for having the condition; NOTE 2: The term is often unclear in lupus anticoagulant testing
because a gold standard test is lacking.
imprecision – dispersion of independent results of measurements obtained under specified conditions;

NOTE: It is expressed numerically as standard deviation or coefficient of variation.
inaccuracy – numerical difference between a measured value and the value of the measurand.
independent test – a lupus anticoagulant test, screening or confirmatory, that is not paired with a test of
the same principle; NOTE: For example, the activated partial thromboplastin time is an independent
screening test and the platelet neutralization procedure is an independent confirmatory test.
indeterminate – not definitely or precisely determined or fixed; not leading to a definite end or result.
index of circulating anticoagulant (ICA) – formula used to calculate a normalized ratio for a mixing
test using either an independent or paired screening test; also referred to as the Rosner Index after the first
author of the original publication.62
integrated test system – a lupus anticoagulant test system that simultaneously incorporates a screening,
confirmatory, and mixing test (no step can be performed independently), eg, the hexagonal phase
phospholipid neutralization test (see Table 3 in Section 15).
laboratory-developed test (LDT) – a class of in vitro diagnostic devices that are manufactured
(including being developed, validated, and offered) within a single laboratory63; NOTE: Laboratories
opting to undertake an LDT should consult appropriate accrediting regulations for guidance on use and
verification of the performance of an LDT.
measurand – quantity intended to be measured (JCGM 200:2012).59
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 (JCGM 200:2012).59
measurement uncertainty//uncertainty of measurement//uncertainty – non-negative parameter

characterizing the dispersion of the quantity values being attributed to a measurand, based on the
information used (JCGM 200:2012)59; NOTE: The parameter may be, for example, a standard deviation
(or a given multiple of it), or the half-width of an interval having a stated level of confidence (ISO
15195).64
measuring interval – set of values of quantities of the same kind that can be measured by a given
measuring instrument or measuring system with specified instrumental uncertainty, under defined
conditions (JCGM 200:2012)59; NOTE 1: In some fields, the term is “measuring range” or “measurement
range” (JCGM 200:2012)59; NOTE 2: The lower limit of a measuring interval should not be confused
with detection limit (JCGM 200:2012).59
mixing – the process by which items are combined or united into one mass; NOTE: The net effect of the
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process is to make the item(s) thinner or more liquid by admixture with something; for example, the
process of mixing a patient plasma with normal pooled plasma results in dilution of each of the
components.
neat plasma – platelet-poor plasma that has not been altered, eg, diluted with normal pooled plasma or
saline.
normal pooled plasma (NPP) – a plasma that is comprised of citrated platelet-poor plasma (with a
platelet count of < 10 × 109/L) from 20 or more carefully screened male and female donors.
normalized ratio – a ratio calculated using clotting times in seconds from an assay in the numerator and
the mean of the reference interval in seconds in the denominator.
paired test system – lupus anticoagulant tests of like principle that are paired as screening and
confirmatory assays, eg, dilute Russell’s viper venom time (dRVVT) screening test and dRVVT
confirmatory test. Results from paired test systems may be expressed as a ratio between the two tests.
(See Table 3 in Section 15.)
platelet-poor plasma (PPP) – centrifuged citrated plasma sample with a platelet count of < 10 × 109/L.
precision (of measurement) – the closeness of agreement between independent test results obtained
under stipulated conditions (ISO 5725-1)61; NOTE: Precision is not typically represented as a numerical
value but is expressed quantitatively in terms of imprecision—the standard deviation or the coefficient of
variation of the results in a set of replicate measurements.
reference interval (RI) – interval between, and including, the lower reference limit (eg, − 2 standard
deviations [SDs]) to the upper reference limit (eg, + 2 SDs) of the reference population; EXAMPLE: The
RI for prothrombin time is 11 to 15 seconds. (See Table 3 in Section 15.)
repeatability (measurement) – measurement precision under a set of repeatability conditions of

measurement (JCGM 200:2012).59
repeatability conditions (of measurement) – conditions where independent test results are obtained
with the same method on identical test material in the same laboratory by the same operator using the
same equipment within a short interval of time (modified from ISO 5725-1).61
reproducibility (measurement) – measurement precision under reproducibility conditions of

measurement (JCGM 200:2012).59
reproducibility condition (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 (JCGM 200:2012).59
responsive – the degree to which the assay is affected by the stated condition.
sample – one or more parts taken from a primary sample (ISO 15189)65; EXAMPLE: A volume of
serum taken from a larger volume of serum (ISO 15189).65
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 (JCGM 200:2012)59; NOTE 1:
Sensitivity of a measuring system can depend on the value of the quantity being measured (JCGM
200:2012)59; NOTE 2: The sensitivity depends on the imprecision of the measurements of the sample.
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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 (ISO 18113-
1).66
trueness (of measurement) – the closeness of agreement between the average value obtained from a
large series of test results and an accepted reference value (ISO 5725-1)61; NOTE: Trueness is usually
expressed numerically by the statistical measure bias that is inversely related to trueness (ISO 17511).60
See also accuracy and bias.
validation – confirmation, through the provision of objective evidence, that the requirements for a
specific intended use or application have been fulfilled (ISO 9000)67; NOTE 1: The World Health
Organization defines validation as “the action (or process) of proving that a procedure, process, system,
equipment, or method used works as expected and achieves the intended result” (WHO-BS/95.1793)68;
NOTE 2: The components of validation are quality control, proficiency testing, validation of employee
competency, instrument calibration, and correlation with clinical findings.
verification – confirmation, through the provision of objective evidence, that specified requirements have
been fulfilled (ISO 9000)67; NOTE: A one-time process completed to determine or confirm test
performance characteristics before the test system is used for patient testing.
4.3 Abbreviations and Acronyms
a activated (ie, IIa, FVa, FVIIa, FVIIIa, FIXa, FXa, FXIa, FXIIa)
AIDS acquired immunodeficiency syndrome
APTT activated partial thromboplastin time
aβ2GPI anti-β2GPI (antibodies against β2 glycoprotein I)
aCL anti-cardiolipin (antibodies against cardiolipin)
aPL anti-phospholipid (antibodies against phospholipid/protein complexes)
APS antiphospholipid syndrome
ASLA activated seven lupus anticoagulant
β2GPI β2 glycoprotein I
BFP-STS biologic false-positive serological tests for syphilis
CAP College of American Pathologists
CEN Comité Européen de Normalisation (European Committee for Standardization)
CV coefficient of variation
dAPTT dilute activated partial thromboplastin time
dPT dilute prothrombin time
dRVVT dilute Russell’s viper venom time
DTI direct thrombin inhibitor(s)
ECAT External quality Control of diagnostic Assays and Tests
ELISA enzyme-linked immunosorbent assay
EQA external quality assessment
F factor (ie, FII, FV, FVII, FVIII, FIX, FX, FXI, FXII)
g gravity
HMWK high molecular weight kininogen
HPE hexagonal phase egg phosphatidylethanolamine
HPNT hexagonal phase phospholipid neutralization test
ICA index of circulating anticoagulant (also called Rosner Index)
IgA immunoglobulin A
IgG immunoglobulin G
IgM immunoglobulin M
INR international normalized ratio
ISO International Organization for Standardization
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ISTH International Society on Thrombosis and Haemostasis

KCT kaolin clotting time
LA lupus anticoagulant(s)
LDT laboratory-developed test
LMWH low molecular weight heparin(s)
NPP normal pooled plasma
PF4 platelet factor 4
PFP platelet-free plasma
PK prekallikrein
PL phospholipid(s)
PNP platelet neutralization procedure
PPP platelet-poor plasma
PT prothrombin time
PT-INR prothrombin time-international normalized ratio
PTT partial thromboplastin time
PTTK partial thromboplastin time kaolin
QA quality assurance
QC quality control
RCPA Royal College of Pathologists of Australasia
RI reference interval(s)
s seconds
SCT silica clotting time
SD standard deviation
SLE systemic lupus erythematosus
SSC Scientific and Standardization Committee
TF tissue factor
TSVT Taipan snake venom time
TT thrombin time
TTI tissue thromboplastin inhibition
UFH unfractionated heparin(s)
UK NEQAS United Kingdom National External Quality Assessment Service
VDRL venereal disease research laboratory
VKA vitamin K antagonist(s)
VTE venous thromboembolism
5 Equipment
Instruments used for clot-based coagulation testing detect a clot end point in several ways. These may
affect routine test results and therefore, potentially, LA testing. Coagulometers may detect a clot by either
of two methods:
 Mechanical—detects physical formation of fibrin strands

 Optical—notes an increase in turbidity with fibrin formation
Either detection method can be used for LA assays, though the sensitivity and specificity of LA screening
tests may be influenced by the type of coagulometer, as shown for dRVVT tests.69 However, threshold
settings for determining a clotting end point on an analyzer have been noted to be more important than the
actual clot detection method.70 With such limited information available concerning instrumentation
effects on LA testing, no analyzer or clot detection end point can be discouraged for use with LA testing.
On the other hand, consideration should be given to using instrument/reagent combinations from one
vendor. In general, if these combinations are available, they may aid in reducing analytical variability.
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6 Validation of Assay Systems

Good laboratory practice along with government directives requires that all tests should be validated by a
laboratory before reporting patient results. These requirements are applicable to all tests used for the
detection of LA. This validation process consists of experiments that define the imprecision, inaccuracy,
reference intervals (RI), cutoff values derived from RI, and interfering substances of each test. In order to
complete an assay validation, a QC program (see Section 14.2) must be initiated to make sure the testing
system is in control and reporting accurate results. The minimally required validation process is that
undertaken for commercially prepared reagents with specific operation (instrument) requirements.
Deviations from a manufacturer’s product insert, modifications of methods, “home-brew” or “in-house”
preparations (also referred to as laboratory-developed tests [LDTs]), or new reagent formulations (relates
to manufacturers) will require additional validation steps. These include more robust RI determination,
assessing analytical sensitivity (limit of quantification), determining analytical specificity (estimating
constant systematic error caused by materials in the specimen that interfere with measurement of the
analyte), and determining any other performance characteristic required for testing (eg, on reconstitution
stability, instrument reagent stability). Once the system is validated, an ongoing QC and QA program (see
Sections 14.2 and 14.3) should be initiated to ensure the accuracy of all test results. Procedures should
also be in place to review the characteristics of any new lot numbers of reagents (see Section 14.1). There
are many guidelines available that provide recommendations on procedures to be used to complete a
method validation (for detailed information, see CLSI documents EP05,71 EP06,72 EP09,73 EP12,74
EP15,75 EP24,76 and H5777).
One special consideration regarding the validation of a new method for LA against a current method is
that for any given set of compared patient samples (given the heterogeneous nature of LA, which may
comprise various mixtures of antibodies for each sample) identical result patterns may not occur. Thus, as
each specific reagent has its own proprietary mixture of components (eg, PL types and concentrations),
reagents may produce varying results with identical patient sample sets. This is supported by EQA data
showing different outcomes in different laboratories testing the same material but using different LA
assays, as well as those using “identical” assays, sometimes associated with testing on different
instruments.78-80 Careful evaluation of comparison data and associated patient results and/or findings is
therefore necessary to fully assess any test under evaluation. All LA assays currently in use do not
directly measure an analyte but instead determine the effect of heterogenous antibodies on a clotting end
point. Therefore, no “gold standard” (international standard) is available to gauge and compare LA
assays.
Validation of methods for LA testing should consider the following:
 Imprecision
 Inaccuracy
 Establishment of RI and cutoff values
 Analytical specificity
 Diagnostic sensitivity and diagnostic specificity
6.1 Imprecision
Precision is described as the closeness of agreement between independent test results obtained under
stipulated conditions, and is not typically represented as a numerical result. Imprecision is the quantitative
assessment of precision, and is usually expressed in the terms of a mean, SD, and CV. In quantitative and
semiquantitative assays, acceptable precision should be demonstrated throughout the measuring interval,
but especially at medical decision levels, eg, around the cutoff value(s) (see CLSI document EP15).75
Parameters that must be measured during the validation of any test include repeatability and
reproducibility. Repeatability (within-run precision) is the agreement between successive measurements
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of the same measurand under the same conditions while reproducibility (total precision) is the agreement
between successive measurements but under changed conditions. Both of these measurements can be
carried out in the same precision evaluation procedure as described in CLSI document EP15.75 Materials
that can be used for these assessments include controls, standards, or patient samples. The material(s)
should mimic the matrix of the samples being tested (see CLSI document EP15).75
For commercial reagents, imprecision data are usually found in the package insert, specific for
reagent/instrument combinations. Experiments used to validate an LA reagent should, at a minimum,
achieve imprecision limits found in the package insert. Reagents that have been modified, used on
instrumentation not provided by the reagent manufacturer, or noncommercially prepared LA reagents
should achieve imprecision limits attainable under similar testing conditions (clot-based assays)
throughout the measuring interval. Refer to CLSI document EP1575 or ISO 5725-1,61 ISO 5725-2,81 and
ISO 5725-382 for experiment design, calculation methods, and criteria for acceptable validation of the
manufacturer’s claims for test repeatability and reproducibility.
6.2 Inaccuracy
When discussing accuracy of a test, results are generally compared to an accepted standard or accepted
value, which equates to “truth.” The lack of an international standard by which to gauge and compare
available assays for LA testing contributes to the challenges for validating LA methods.
Although establishing the existence of identical data between compared methods may be elusive,
assessment of like-to-like assays (eg, one dRVVT to another dRVVT) should produce broadly similar
results, particularly those represented by clearly normal and abnormal samples. Lack of correlation
between reagents and/or instrumentation is more likely observed for borderline LA samples. Verification
of commercial methods by manufacturers should similarly focus on such comparative evidence.
Initial determination for a new method to demonstrate trueness or measure of bias between methods
should include a minimum of 40 samples that span across the entire measuring interval (LA not detected
and LA present, including borderline and strongly positive samples). A reagent lot conversion requires
only 20 samples that span the measuring interval (normal, abnormal, and samples around the cutoff
value). If reagent comparison is required, then testing should be performed on the same analyzer using
both the current and new reagent. If instrumentation comparison is required, then testing should be
performed using the same reagent on both the current and new analyzer. Samples used for comparison
studies could include any of the following:
 Properly stored and well characterized LA samples

 Commercially available material with LA results
 “Split samples” that have been sent to an accredited reference laboratory for clinical testing
Samples should be tested using the same aliquot maintained under similar conditions (eg, fresh vs fresh or
frozen vs frozen), and, if frozen, from the same freeze/thaw cycle. Testing should be performed on five to
eight samples per day and take place over five to eight days in order to include the inherent differences in
reagent vials, technologists, and analyzer performance. Testing should be performed according to
recommendations from both the instrument and reagent manufacturer. If duplicate testing is
recommended, then CLSI document EP0973 must be followed regarding the order of testing for duplicate
samples. If samples are not of adequate volume to perform the method comparison or duplicate testing
with each method, then minipools can be created by pooling together two patient plasmas that have
similar results. No more than two samples should be included in this minipool in order to maintain
individual sample matrix (see CLSI document EP0973).
Acceptance criteria for demonstration of trueness by comparison of samples can be found in CLSI
document EP15.75 These criteria include statistical and graphic representations of data. For convenience,
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computer software is also available for determining acceptability of patient comparison studies using
CLSI guidelines. In addition, a 2 × 2 truth table should be evaluated to determine whether differences seen
in numerical data significantly affect diagnostic sensitivity (see Section 6.5).
6.3 Establishment of Reference Intervals and Cutoff Values
Establishing an RI is important for all clinical laboratory assays because this range provides an estimate
regarding what is considered normal in the overall population. Moreover, for LA tests, the upper limit of
normal determines the cutoff value for tests while the RI mean is used for normalizing LA assays. An RI
(range) flanked by its lower and upper limits is determined using statistical tools and is associated with a
degree of uncertainty. Refer to CLSI document EP2883 for specific guidance and in-depth details of the
many issues associated with determining an RI. An RI needs to reflect the patient population (pediatric,
obstetrics, geriatric, etc.) served by the site.
A laboratory must establish an RI for each LA test as well as any associated calculations (normalized
ratios, percent correction, index of circulating anticoagulant [ICA], etc., as noted in Appendix B) used in
conjunction with an LA test. This guideline recommends that samples from at least 40 normal, healthy
subjects (as defined in CLSI document EP2883) be used for establishing an RI. Additionally, these
samples must be platelet poor (< 10 × 109/L) and, if possible, reflect how patient samples are tested. For
example, if testing fresh patient samples, then use fresh normal samples for establishing an RI; or, if using
frozen patient samples, use normal samples that have been frozen and thawed for establishing an RI.
Reagents used for establishing an RI should be used in the same manner they are used when patient
samples are tested. Once data have been collected, they must be verified using a histogram to ensure that
data points are distributed in a gaussian fashion. If outliers are present, refer to CLSI document EP2883 for
detailed information regarding their inclusion or exclusion. With normal distribution confirmed and
reconciliation of outliers completed, data should be analyzed using parametric statistics to calculate a
mean and ± 2 SD interval. The + 2 SD number serves as the cutoff. Appendix B can be used as a template
for establishing an RI and the supporting statistics can be calculated using spreadsheet programs.
This guideline aligns with CLSI document EP2883 but does not align with the ISTH SSC 2009
guideline,28 which recommends that cutoff values be set at the 99th percentile. Percent or quantile
distribution is not readily determined using parametric statistical calculations and is therefore not
recommended by this guideline. The use of ± 2 SDs is supported by publications indicating that data,
collected for clot-based assays such as the PT and APTT, show a normal gaussian distribution.84-87 Many
LA assays are based on the APTT or PT; thus, use of normally distributed data can be extended to these
tests. Gerbutavicius and colleagues support this position as data derived from ≈ 90 healthy subjects were
normally distributed for LA assays such as the TTI and dRVVT. Furthermore, they showed that RI for
these LA assays were tighter (less broad) when parametric vs nonparametric statistics were applied.88
No statistical tool is perfect and this guideline accepts that limitations exist with using parametric
statistics for establishing an RI. A ± 2 SD RI will yield “abnormal” results for 5% of the “normal
population” (only 95% are included as normal, leaving 2.5% at either “tail” to potentially yield an
abnormal test result). Statistically, this percentage (2.5%) may even be higher because confidence
intervals around this upper limit may not be robust. Hence, the potential for false-positive results exists.
However, as noted in Section 13, it is not individual tests but composite LA testing that will be used to
make a laboratory diagnosis of LA.
Under some circumstances, a laboratory does not need to establish an RI but may transfer an established
RI for its own use. An RI established by a manufacturer can be transferred for a defined
instrument/reagent combination. This option requires that manufacturers have well-established RI and
cutoff values (mean with ± 2 SDs) using a minimum of 120 normal samples (or more per a manufacturer’s
verified method) and, if queried, provide details of RI studies (ie, number of reference subjects and their
demographic information, inclusion and exclusion criteria, reagent lot number and instrument type, and
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the statistical method used for calculation). RI can also be transferred from one laboratory to another as
long as the same reagents are used and the same instrumentation shares identical programming/analytical
protocols. This document does not support transference of an RI when changing from one instrument to
another or when changing from one reagent to another. Within a laboratory, once an RI for a specific
method has been established, it can potentially be transferred when, for example, changing from an old to
a new lot number of reagent.
CLSI document EP2883 outlines several ways in which a receiving laboratory can challenge and thereby
accept a donor laboratory’s RI or accept a previous RI when changing lot numbers of reagent within a
laboratory. This guideline recommends that the verification method using a minimum of 20 reference
individuals be followed. If the process is followed as recommended in CLSI document EP28,83 then the
RI in question may be successfully transferred. A concern with the transference is that there is little or no
published information supporting this practice with LA assays.
6.3.1 Normalization of Lupus Anticoagulant Screening and Confirmatory Test Results
Use of normalized ratios is recommended for expressing LA results to reduce the effects of inter- and
intra-assay variation.28,29,89 This guideline recommends the use of normalized ratios for reporting test
results from LA screening and confirmatory assays (with the exception of LA assays that report results as
delta values). Normalized ratios are derived by placing the patient’s test result (time in seconds) in the
numerator and the mean of the RI (time in seconds), for the specific test performed, in the denominator.
Ratios are reported to two decimal places. RI for normalized ratios are established subsequent to
establishing an RI for each LA screening test and confirmatory test. Each of 40 normal sample test results
is divided by the mean RI value of a specific test and thereby generates a normalized ratio for each
normal donor. A mean and ± 2 SD interval is determined using the 40 sample ratios and the + 2 SD
number serves as the cutoff value (see Appendix B).
For generating a normalized ratio, two values have been used for the denominator:
 The mean clotting time of the RI for a specific assay69,90-93

 The clotting time of the normal pooled plasma (NPP) analyzed with each batch28,29
This guideline does not support the use of the NPP in the denominator because not all NPP will generate
the same clotting times with the various reagents (numerous vendors) used for the same test type (eg,
dRVVT).86,94 Furthermore, results from NPP preparations may not correlate with those of patient samples
if NPP is lyophilized or NPP and patient samples are collected into differing trisodium citrate
preparations.95-97 Use of the RI mean negates the variability encountered between NPP preparations and
batches.98 The RI mean should also compensate for inter- and intra-assay variability
(operator/reagent/analyzer) because these issues are considered in the process of establishing an RI. (For
detailed information, see CLSI document EP28.83) Currently, several reagent manufacturers support the
use of the mean RI in their product inserts. Use of the mean of the RI predicates that RI will be
established/verified with each new lot of reagents; otherwise, the use of an “old” RI with “new” reagents
will not permit this compensation in day-to-day variation.
6.4 Analytical Specificity
Analytical specificity estimates potential constant systematic error caused by materials in the specimen
that may interfere with measurement of the analyte, regardless of analyte concentration. Common
interferences when using clot-based assays are the presence of small clots, bilirubin, hemolysis, and
lipemia, of which all or some may have an impact, in part depending on the type of instrumentation used.
Anticoagulants such as heparin (unfractionated [UFH] or low molecular weight [LMWH]), vitamin K
antagonists (VKA), direct thrombin inhibitors (DTI), and inhibitors (direct or indirect) of activated FX
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may affect clot-based testing. UFH can prolong APTT-based, PT-based, and dRVVT assays. The
interference is greater with APTT-based because many PT reagents and kit-based dRVVT reagents
contain heparin neutralizers. LMWH preparations, which inhibit activated FX and activated FII
(thrombin), may differ in the ratio of anti-Xa to anti-IIa activity and thereby affect LA testing.28 A
synergistic effect can result if LMWH and a borderline (weak) LA are both present. The combined effect
could prolong the APTT sufficiently to yield abnormal LA test results. VKA cause an acquired factor
deficiency of the vitamin K–dependent FII, factor VII (FVII), factor IX (FIX), and FX. Refer to the
limitations section for each test, to Section 7.1.3, and to Appendix E for more detailed information
regarding the effects of pharmacological agents on LA testing. Certain hereditary or acquired
coagulopathies may also have the potential to yield false-positive LA results. This outcome has been
reported with both factor VIII (FVIII) and factor V (FV) inhibitors.99,100 With contact factor deficiencies,
the APTT is prolonged (the degree depends upon reagent sensitivity to contact factor deficiencies) and,
similarly, APTT-based LA assays such as the PNP and HPNT may give rise to spurious confirmatory test
results.
Most manufacturers’ product inserts state which agents or coagulopathies may interfere with testing.
Once RI and cutoff levels have been defined for each LA test, it is advisable to challenge these limits with
plasma samples containing interfering drugs and known coagulopathies, if available.
6.5 Diagnostic Sensitivity and Diagnostic Specificity
Diagnostic sensitivity defines the ability of a test to correctly identify a disease (true positive) at a
particular decision threshold, whereas diagnostic specificity defines the ability of a test to correctly
identify the absence of a disease (true negative) at a particular decision threshold. Diagnostic sensitivity
and specificity are determined by challenging RI/cutoff limits that have been established for each LA
assay. To determine diagnostic sensitivity, abnormal LA samples (samples that are borderline [near cutoff
values] and samples showing LA presence), which have been confirmed previously in one’s own
laboratory, obtained commercially, or from another laboratory, are used. To determine diagnostic
specificity, samples should be obtained (from sources as noted above) from normal individuals (not the
same individuals used to establish/verify the RI) and from patients for whom LA is not detected but
whose plasmas contain some interfering substance. These latter representative samples may include
interferences by UFH, LMWH, VKA, DTI, pharmacological inhibitors of activated FX, FVIII inhibitors,
etc. Details of an evaluation process to define these parameters are available in CLSI document EP24.76
However, the process is challenging in LA testing, due to the heterogeneity in clinical symptoms
associated with the “diseases” for which LA testing may be employed, and because there is no gold
standard assay that can be used to define a true test positivity.
The lack of a gold standard assay suggests that false-positive results (well persons are misclassified as
having a disease) and false-negative results (diseased persons are misclassified as well) do not exist with
LA assays. However, as noted in Section 6.4, many pharmacological agents and coagulopathies may
interfere with specific LA assays. These interferences may falsely prolong a clotting time and suggest the
presence of the inhibitor (false-positive result) or may affect testing to shorten clotting times and mask the
inhibitor (false-negative result). Misclassifications may also occur with inappropriate cutoff values. If
determined cutoff values are set too high, the risk of false positivity is reduced but there is a risk that
false-negative results may be obtained.101,102
7 Preexamination Issues (Criterion A)

Preexamination issues relate to specimen collection, transport, centrifugation/processing and storage, and
to patient-specific issues including anticoagulant therapy, acute phase reactions, and underlying
conditions. Guiding clinicians in appropriate test ordering can play a useful role at this initial phase of
testing (such as avoiding testing during anticoagulant therapy).
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Clinicians may request a routine coagulation screen (APTT and PT) in asymptomatic patients before an
invasive procedure to exclude significant hemostatic abnormalities. The validity of this type of
preoperative testing is beyond the scope of this document. However, if a prolonged APTT is identified
and is unexplained, a reason should be found before the procedure, to determine if LA, an APTT
detectable factor deficiency, or specific factor inhibitor is present. The distinction is important because
patients with LA may be at risk for thrombosis (although patients can also be asymptomatic), whereas
factor deficiencies and specific factor inhibitors can increase the risk for bleeding, depending on the
deficient factor.
7.1 Patient Selection
Defining the appropriate patient for LA testing may be predicated on:
 Finding an abnormal laboratory screening test (APTT, PT), in the presence or absence of
thrombophilia
 Evaluating patients with venous or arterial thrombosis or certain types of pregnancy morbidity
Therefore, the physician and his/her ordering practices play a role in the preexamination phase.
An update from the ISTH SSC introduced three categories of patients, based on probability of having
APS, for whom LA testing may be appropriate.28
High-probability patients have:
 Unprovoked venous thromboembolism (VTE) or unexplained arterial thrombosis younger than age 50
 Thrombosis at unusual sites
 Late pregnancy loss
 Any thrombosis or pregnancy morbidity in the setting of autoimmune diseases (SLE, rheumatoid
arthritis, autoimmune thrombocytopenia, autoimmune hemolytic anemia)
Moderate-probability patients have:
 An incidental APTT prolongation

 Recurrent miscarriage early in gestation
 Provoked VTE in young patients
Low-probability patients have:
 VTE or arterial thrombosis at an elderly age
Testing for LA in patients other than those listed above is discouraged.28
7.1.1 Patients With Abnormal Routine Coagulation Tests in the Absence of Clinical
Manifestations
Historically, the prolongation of an APTT in patients with an absence of bleeding was a primary
consideration for LA testing. Because there is significant variability of APTT and PT commercial
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reagents to the LA effect, relying strictly on the routine APTT and prothrombin time-international
normalized ratio (PT-INR) as the initial component of a diagnostic algorithm is not suitable.
For routine APTT testing (general coagulation testing), the majority of clinical laboratories in the United
States have reported using APTT reagents that are designed or modified to detect LA.103 Because APTT
prolongations are not specific for LA, this would mean that all asymptomatic (eg, no evidence of arterial
or venous thrombosis) patients tested in the clinical laboratory are being unnecessarily “screened” for the
presence of LA, which is contrary to current recommendations and is discouraged by this guideline and
the ISTH SSC guideline.28 Therefore, it has been suggested that laboratories, for routine clinical use,
avoid using APTT reagents that are designed to increase detection of LA.104,105
The varying degree to which commercial APTT reagents detect LA has been described.106,107 Participants
in a College of American Pathologists (CAP) survey used a spectrum of APTT reagents, which
demonstrated a variety of different results to LA abnormal samples.106 It has been shown that commercial
reagents for PT-INR testing also are variably affected by LA,108-110 with certain recombinant tissue factor
(TF) reagents having the highest responsiveness to LA.111 Therefore, using only a routine APTT to screen
for LA would be insufficient. It is plausible that a given laboratory may use APTT reagents that are less
affected by LA, while using a recombinant PT reagent, which may be more affected. Laboratory
personnel should be cognizant of the degree to which their reagent/instrument systems are affected by LA
and consider LA testing in patients with unexpected prolongation of the APTT and/or PT-INR.
In patients with unexplained acquired hypoprothrombinemia (FII deficiency), LA testing may be

warranted, because prothrombin antibodies associated with acquired hypoprothrombinemia and LA have
been described. These antibodies against prothrombin are typically non-neutralizing, allowing a
correction of mixing tests, erroneously suggesting the absence of an inhibitor such as an LA.112,113 In
contrast, there are antibodies against prothrombin with inhibitory properties that confer LA
activity.18,113,114
LA can also rarely cause immune-mediated transient FX deficiency and bleeding, with clinical and
laboratory findings similar to LA-induced hypoprothrombinemia, except that FX is depleted by non-
neutralizing LA-related antibodies.115 Immune-mediated protein S deficiency, associated with LA, and
complicated by thrombosis or purpura fulminans, has been described in children in association with
preceding infections116 and in patients with SLE.117
7.1.2 Patients With Suspected Antiphospholipid Syndrome
APS should be considered in patients with vascular thrombosis or certain types of pregnancy morbidity.2
Patients with objective diagnostic evidence of arterial, venous, or small vessel thrombosis (not including
superficial thrombosis) should be considered for LA testing. Objective diagnostic studies for thrombosis
include angiography, computerized tomography, venography, compression ultrasound, or histological
evidence of thrombosis without vessel wall inflammation. Other features that may be associated with APS
include cardiac valve disease, neurological changes, livedo reticularis, nephropathy, and
thrombocytopenia, but these clinical manifestations do not meet the clinical classification criteria for
APS. Females with a history of pregnancy morbidity (≥ 1 unexplained fetal death with normal
morphology ≥ 10 weeks of gestation; ≥ 1 premature delivery of a newborn with normal morphology
before the 34th week of gestation, secondary to eclampsia/severe preeclampsia or placental insufficiency;
≥ 3 consecutive spontaneous abortions before 10th week of gestation, with no chromosomal [fetus and/or
both parents] or maternal anatomic or hormonal abnormalities) should be considered for LA testing as
part of the laboratory criteria (along with IgG and IgM aCL and/or aβ2GPI) required for diagnosis of
APS.2,118
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7.1.3 Anticoagulant Therapy
Ideally, patients should not be receiving anticoagulant therapy at the time of testing. UFH, if not removed
before testing, may produce false-positive LA screening tests (false prolongations) and mixing tests (false
noncorrections), and may complicate interpretation of confirmatory assays.119 Some LA reagents contain
a heparin neutralizer, which can neutralize a specified amount of UFH in the specimen. Results should be
interpreted with caution, because false results may be obtained if the amount of heparin in the specimen
exceeds the heparin-neutralizing capability of the reagent. The effect of VKA such as warfarin on LA
tests is not well studied, but warfarin prolongs the PT, APTT, and dRVVT, which can make interpretation
of results challenging. In addition, if the prolongations are significant, false-positive (noncorrecting)
mixing tests can be obtained.
DTI (eg, argatroban, bivalirudin, hirudin, dabigatran) can cause false-positive (noncorrecting) mixing
tests and, in some instances, false-positive confirmatory tests. Mechanisms to check for the presence of
DTI include the thrombin time (TT), which would be prolonged but subsequently not shortened by
heparin neutralization (see Section 8.3), or checking the patient’s medications in their medical record.
There is limited information regarding the effects of fondaparinux or rivaroxaban, inhibitors of activated
FX on LA testing, but those agents will also lead to noncorrection of mixing tests. Refer to the limitations
sections for specific tests and to Appendix E for more detailed information regarding the effects of
pharmacological agents on LA testing.
7.1.4 Biological Variability (Acute Phase Reactions)
Acute phase reactions have the potential to interfere with LA testing, because FVIII and fibrinogen
become elevated during an acute process and thereby shorten the APTT and other clotting times. Elevated
FVIII or short baseline clotting times might cause false-negative LA tests by preventing what would
otherwise be a prolonged clotting time.101,120-122 Pregnancy creates a hypercoagulable state in the patient in
part by raising fibrinogen and FVIII, which could shorten clotting times. Fibrin/fibrinogen degradation
products may prolong clotting times, whereas microparticles from activated platelets may shorten clotting
times or bypass LA activity, thereby shortening prolonged clotting times. Organic elevations of C-
reactive protein may lead to false-positive results as shown in a study wherein spiking C-reactive protein
to levels > 53 mg/L in vitro caused false-positive HPNT results, and levels > 24 mg/L caused an elevated
normalized APTT ratio. dRVVT results were not affected.123
7.1.5 Patients With Systemic Lupus Erythematosus
In 1982, the American College of Rheumatology revised the diagnostic criteria for the classification of
SLE.124 These diagnostic criteria include clinical and laboratory features. The SLE classification criteria
were modified again in 1997, with the immunological component requirement changing from positive
lupus erythematosus preparations to positive aPL, which included IgG or IgM aCL and/or LA.125,126 The
most recent guidelines, published in 2012, also include elevated IgA β2GPI antibodies and moderately or
markedly elevated IgA aCL in the diagnostic criteria.127 LA has a prevalence of ≈ 25% in the SLE
population,128 associated with an increased risk for arterial and venous thrombosis, as well as myocardial
infarction in the adult and pediatric populations.128-130 While it is generally accepted that LA testing is part
of the diagnostic criteria for SLE, the frequency of testing in seronegative patients remains unclear. It is
also unclear whether or not repeat testing in patients with positive LA is required.130,131
7.2 Specimen Collection and Transport
Samples for the detection of LA must be collected in the appropriate specimen collection tube (sodium
citrate) (see CLSI document H21132). Aliquots processed at referral sites should be labeled with
anticoagulant type to prevent LA testing on inappropriate samples (ie, EDTA, serum), which can lead to
false-positive testing.133
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7.2.1 Phlebotomy
Specimens should be collected into tubes commonly described as 3.2% sodium citrate (105 to 109
mmol/L, 3.13% to 3.2% of the dihydrate form of trisodium citrate [Na3C6H5O7 • 2H2O]) following the
procedure outlined in CLSI documents GP41134 and H21.132 Samples should be rejected if they are not
collected in sodium citrate, are under- or overfilled when collected in sodium citrate, or show evidence of
clots (see CLSI document H21132). Special attention should be given to the draw order of the tubes to
ensure the light blue top (sodium citrate) is drawn before any “activating” tubes (eg, thrombin-activating
tubes) that may result in in vitro clot formation, and before tubes containing any other anticoagulants
(EDTA or heparin) that may result in sample contamination. Tubes should be mixed by gentle multiple
inversions immediately after drawing the specimen to ensure the blood and anticoagulant are mixed well.
Specimens should not be shaken vigorously. Proper collection and mixing will prevent in vitro clot
formation and activation of platelets, which may cause false-negative LA testing results.
CLSI documents H47,135 H21,132 and GP41134 indicate that if only a light blue top tube is collected, no
discard tube is required. However, there are no specific data about single draw collection technique and
the effect on LA tests using standard evacuation tube systems. If the blood specimen is procured with a
winged needle collection system, the first tube will underfill, and therefore a nonactivating priming
(discard) tube is required before sample collection for LA testing (see CLSI document H21132 for details).
Proper filling of the sodium citrate tube (ratio 9:1 [blood: anticoagulant]) is essential for obtaining correct
results, and manufacturers’ recommendations on fill limits should be followed. Variances to this ratio
such as underfilling (short draw) of collection tubes or an elevation in patient hematocrit (anticoagulant
volume should be adjusted if > 0.55) (see CLSI document H21132 for details) can cause prolongation of
clot-based tests, which could appear similar to an LA. This prolongation is caused by excess sodium
citrate in plasma that complexes to calcium introduced into an assay (recalcification) and thereby makes
available less calcium to promote clotting in a test (clotting process is slowed and times are prolonged).
7.2.2 Transport to Laboratory
Screening tests for LA are commonly based on the APTT, dRVVT, or PT, so specimens should be
processed within four hours of collection (see CLSI document H21132). It is recommended that capped
whole blood and platelet-poor plasma (PPP) (< 10 × 109/L) specimens/samples for coagulation tests should
be transported to the laboratory at room temperature (18 to 24°C) as described in CLSI document H21.132
Though storage recommendations for PPP to be used for LA testing are not specifically addressed, CLSI
document H21132 states that it is acceptable for assays other than PT or APTT to be stored at room
temperature (18 to 24C) or refrigerated (2 to 4C) for a maximum of four hours.
If a specimen is sent to the laboratory by pneumatic tube, the specimen should be wrapped to prevent
excessive agitation and activation of platelets. Specific data about the effect of pneumatic tube systems on
LA testing are limited, and this recommendation is conservatively based on specimen/sample transport
recommendations for PT and APTT (see CLSI document H21132 for details).
7.3 Sample Preparation
It is critical for testing accuracy that specimens collected and tested for LA are processed in a manner that
will result in plasma that is platelet poor (< 10 × 109/L). Residual platelets contain PL in the cell
membrane that may neutralize LA. They are more problematic with a frozen sample that has been thawed
because platelets will burst, releasing excess PL into the plasma.136 This increase in PL content may cause
a false-negative test by shortening clotting times.28
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7.3.1 Centrifugation for Preparation of Platelet-Poor Plasma
PPP is used for LA testing in clinical laboratories. PPP contains residual platelets with a recommended
platelet count of < 10 × 109/L. Ideally, the PPP is double-centrifuged using a swing-out bucket centrifuge
to ensure minimal residual platelets in the plasma used for LA testing. In the clinical laboratory, the use
of filters to obtain PPP is not recommended. Cellulose acetate filters, specifically, may cause binding of
factors (FV, FVIII, FIX, factor XII [FXII] and von Willebrand factor) to the membrane, causing increased
clotting times.133 Preliminary work with low protein binding filters has been encouraging, but in the
absence of published evidence, this document cannot recommend their use. Platelet-free plasma (PFP),
which is devoid of platelets, can be prepared from PPP using a combination of ultracentrifugation and
microfiltration.137 Ultracentrifugation, however, can generate microparticles that interfere with LA
testing.138
PPP is obtained by centrifugation at a speed and time that results in a residual platelet count of < 10 ×
109/L. This can be accomplished by centrifuging specimens at 1500 gravity (g) force for no less than 15
minutes at room temperature. In order to minimize remixing of plasma and red blood cells, a swing-out
bucket rotor should be used and the brake not applied at the end of centrifugation (see CLSI document
H21132). Plasma should be carefully separated from the buffy coat with a plastic, disposable pipette and
aliquots approximating 1 mL transferred to polypropylene microtubes (preferred), minimizing dead space,
and capped. Residual platelet count of PPP can be verified, at least annually, using an automated cell
counter (see CLSI document H21132).137 It is critical that NPP, used for mixing tests in various LA
screening/confirmatory assays, also meets the recommended residual platelet count.
If a residual platelet count of < 10 × 109/L is not achieved by centrifugation as noted above, then double
centrifugation should be performed (see Figure 1).139 The original specimen tube is centrifuged as stated
above. Plasma from this original tube is carefully removed with a plastic disposable pipette without
disturbing the buffy coat/platelet layer and transferred into a nonactivating (eg, polypropylene or similar)
plastic tube. This second tube is capped and centrifuged again at 1500 g for 15 minutes at room
temperature. Plasma from this second tube is again carefully removed, as noted above, and transferred to
a polypropylene tube. Polystyrene tubes may crack if frozen below −30°C and should be avoided if
samples are to be stored at or below this temperature (see CLSI document H21132).28 The platelet count of
PPP obtained in this manner should also meet the target count of < 10 × 109/L.
1. Centrifuge the capped specimen tube.
2. Remove plasma layer and transfer to an aliquot tube, being careful not to disturb the buffy coat (white
blood cells and platelets).
3. Cap and centrifuge the first aliquot tube.
4. Remove plasma, leaving a small amount at the bottom of the tube and using care not to aspirate the
small pellet of red blood cells and platelets at the bottom.
5. Transfer this PPP to a second clean polypropylene aliquot tube, cap, and either use or freeze.
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Plasma
Buffy Coat
1 Red Blood Cells

A
2 3 A B
4 5
Figure 1. Double Centrifugation Procedure
There are no published recommendations on how to handle multiple specimen tubes from the same
patient. If multiple specimen tubes are collected from one patient, PPP from each centrifuged specimen
tube can be transferred to individual polypropylene tubes, or, alternatively, pooled together and then
aliquotted into polypropylene tubes. The latter choice is discouraged because plasma from a poor-quality
specimen tube (eg, a tube with clots) could be mixed with others that were properly collected, thereby
contributing significantly to a preexamination error. It is imperative that each individual tube is checked
to make sure a specimen is not clotted.
Samples that appear hemolyzed, lipemic, or icteric do not present an immediate cause for specimen
rejection. Hemolyzed or icteric samples can be tested with nonphoto-optic systems (eg, mechanical clot
detection) or with photo-optic clot detection systems, which use wavelengths insensitive to these
interferences. Though analyzers may not be affected by or compensate for hemolysis, the quality of these
specimens should be questioned because hemolysis can cause potential activation of the clotting
system.140 Samples that are lipemic or icteric may interfere with analyzers that use light-scattering clot
detection methods. These analyzers tend to “flag” results that do not meet instrument-defined clot curve
criteria, suggesting invalid or suspicious results.
7.3.2 Storage of Sample Aliquots
Specific data about the effect of storage conditions on LA testing are limited, and the following
recommendations are conservatively based on storage recommendations for PT and APTT (see CLSI
document H21132). Manufacturers’ package inserts should be consulted for proper storage of samples
specific to their test system.
Samples tested within four hours of collection may be kept at room temperature if capped. If specimens
are uncapped, prolonged exposure of plasma to air may result in CO2 release and thereby increase the pH
of the sample, which may falsely prolong PT- and APTT-based tests.137
Samples not tested within four hours of collection should be stored in polypropylene tubes and frozen.
Woodhams et al. have studied the stability of coagulation test results on samples stored in a 5-mL tube
and a screwtop micro-vial to determine the variability of results over time. They determined that the
screwtop micro-vial with a small volume produced the least variability in coagulation results, especially
when stored at −70C.141 Plasma tubes should be quickly frozen at −70C or colder.28 Plasma is stabile at
this temperature for at least six months (see CLSI document H21132).
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If a −70C freezer is not available, plasma can be stored in a −20C freezer. The use of a freezer that
undergoes automatic freeze/thaw cycles (ie, frost-free freezers) is not recommended unless it has a
continuous-monitoring temperature recording device, or a minimum-maximum thermometer. The
acceptable temperature range for a frost-free freezer must not be exceeded because temperature deviation
can cause factor deterioration and cold activation of FVII. Plasma is stabile for 14 days when stored
appropriately at −20C (see CLSI document H21132).
7.3.3 Thawing of Samples Before Testing
Samples removed from the freezer should be immersed in a 37C water bath, for approximately five
minutes, to completely thaw the plasma and avoid denaturing fibrinogen. Samples must be thoroughly
mixed by gentle multiple inversions before testing and LA tests must be completed within four hours of
thawing.28,137
Samples stored in dry ice (solid CO2) containers should be thawed uncapped in a 37C dry bath
(incubator) for at least 15 minutes to allow equilibration with ambient air and evaporation of plasma
CO2.142 If CO2 is retained in the plasma, it can cause spuriously prolonged PT clotting time test results
(and by extension, possibly LA test results) because of an altered plasma pH.143
8 Preliminary Examination Issues

Routine assays such as the APTT, PT, and TT may be the first tests used by a general coagulation
laboratory that may lead to either a suspicion that LA is present or to the evaluation of a sample for the
presence of LA. Not only can these tests aid in screening for LA, but they also provide information
regarding the integrity of a sample and assist in determining the presence of pharmacological agents that
may affect further testing and LA interpretation.
Results of routine tests, eg, the APTT, and less commonly the PT, can be elevated in the presence of LA.
Therefore, it is important for a laboratory to fully understand the impact that LA may have on its routine
reagents. Depending on the need of a laboratory, APTT reagents should be evaluated and selected for
their responsiveness or lack thereof to LA. PT reagents may also be affected by LA and this should be
considered when using this test for monitoring therapy with VKA. A prolonged TT is one of the best tests
for determining the presence of heparin or DTI. Alternatively, a heparin assay or heparin neutralizers
(heparinase or polybrene) can be used to determine if heparin is present.
8.1 APTT
The APTT is a routine global coagulation test procedure expected to perform three specific functions:
 Identify quantitative and qualitative factor abnormalities in the intrinsic and common pathways of
coagulation that may be associated with bleeding risk.
 Monitor therapy with UFH or certain other anticoagulants.
 Detect inhibitors of blood coagulation (eg, LA).7,144
All three conditions should cause a prolongation of the APTT, the degree to which is dependent upon the
responsiveness of the reagent to factor deficiencies, UFH, or LA. These three functions for an APTT
reagent drive the manufacturing of these products, demand that laboratories know the needs of their
patient populations, and require laboratories to evaluate the responsiveness of reagents for these intended
uses. The composition of APTT reagents, as noted below, varies significantly, thereby impacting how an
APTT reagent (or lot of an APTT reagent) will perform.145 CLSI document H47135 details how
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laboratories can evaluate APTT reagents for responsiveness to factor deficiencies (factor sensitivity
curves) or presence of heparin (heparin sensitivity determination using anti-Xa assays). In contrast,
sensitivity of an APTT reagent for LA cannot be quantified. As outlined in Section 6.2, laboratories must
determine the impact that the presence of LA (using samples that are either clearly abnormal or
borderline) will have on APTT clotting times.
The PTT was described by Langdell, Wagner, and Brinkhous in 1953.146 In 1961, Proctor and Rapaport
modified the PTT by adding an activator, kaolin, based on the studies by Margolis.147 Thus, the APTT
was born (see Appendix D, Figure D1) and continues to the present day as a mainstay in coagulation
laboratories worldwide. Fundamentally, an APTT reagent is composed of:
 An activator, which converts FXII to its activated form

 PL, which replaces in vivo platelet PL surfaces, on which coagulation reactions occur
 Buffer, which minimizes pH changes in plasma reaction mixture
CaCl2 is added after the activation (incubation) period in order to permit FIX activation by activated
factor XI (FXIa) and drive the coagulation cascade to completion. CaCl2 reintroduces calcium ions that
were chelated by sodium citrate anticoagulant in the blood collection tube.
APTT reagents differ in:
 Types of products used to activate the contact system, which are particulate activators, such as
micronized silica, colloidal silica, kaolin, and ellagic acid148
 Source material for PL such as rabbit brain, bovine brain, placenta, purified soybean, or synthetic
lipidation in liposomes
 Type(s) of PL that are present in the source material (eg, phosphatidylinositol, phosphatidylserine,
phosphatidylethanolamine, and phosphatidylcholine)
 Percent concentrations of some combination of the above PL in the reagent milieu
 Configuration of PL such as liposomes, micelles, or protein-bound, all of which determine how PL is

presented to coagulation proteins
How the above differences are exploited for manufacturing APTT reagents is proprietary knowledge and
unique to each vendor and each reagent. Laboratories should select APTT reagents to evaluate and
implement according to the needs of their institution, but using APTT reagents that are responsive to LA
is not recommended for screening the general population (see Section 7.1). Examples that may benefit
from using APTT reagents that are minimally responsive to LA (ie, the antibodies rarely prolong the
APTT above the upper limit of the RI) include:
 A pediatric hospital in which slightly elevated APTT values due to transitory, infection-induced aPL
may cause further, expensive, and unnecessary testing to be performed or surgeries postponed
 A laboratory that only screens for bleeding disorders
 A laboratory wishing to reduce interference by the antibody when performing factor assays on
samples that are positive for LA
 A facility that only monitors heparin therapy144
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Laboratories may choose to have more than one type of APTT reagent in stock for testing (eg, LA-
responsive reagent and a reagent that is minimally affected by LA).
The degree to which APTT reagents are affected by LA rests predominantly with the concentration of PL
in the reagent; ie, increasingly higher, though not defined, levels mitigate the effect of LA. 149
Furthermore, reagents rich in phosphatidylserine eliminate the LA effect on the APTT150; hence, APTT
reagents that are modified to have lower PL content and lower percentages of phosphatidylserine are
better suited for detecting LA.151 Therefore, the class, concentration, and conformation of PL play a role
in the detection of LA.100,152 In general, in the presence of LA, a reagent that is less responsive to LA
yields shorter clotting times than reagents that are affected by LA.153 Manufacturers offer APTT reagents
containing low concentrations of PL that are specifically designed for detecting LA (see Section 10.2).
Any APTT reagent, irrespective of its responsiveness to LA, may be affected by the presence of acute
phase reactant proteins such as FVIII and fibrinogen.154 For this reason, a normal APTT, even when using
reagents modified to detect LA, does not exclude LA.
8.2 Prothrombin Time-International Normalized Ratio
The PT is a screening coagulation assay that assesses the function of FVII, FX, FV, FII, and fibrinogen. It
is performed as a one-step reaction that was first described by Quick.155
Thromboplastin or PT reagent is added to a patient’s sodium citrate plasma and the time in seconds for
clot formation is determined (see Appendix D, Figure D8). Commercial thromboplastin reagent contains:
 TF (initiates coagulation) sourced as recombinant human TF or from extracts of bovine brain, rabbit
brain, or human placenta
 PL (required for surface assembly of coagulation macromolecular complexes)
 A physiological concentration of calcium chloride (reintroduces calcium ions that were chelated by
sodium citrate anticoagulant and is necessary for proper orientation of proteins and their binding to
PL)145
Commercial PT reagents vary in their responsiveness to LA but most are not responsive because PL differ
in type or are at higher concentrations than those found in APTT reagents.89,91,108,156 Rabbit brain and
recombinant human tissue thromboplastins are more responsive to LA than bovine brain
thromboplastin.110,157 Many commercial PT reagents contain a heparin neutralizer that, in general,
neutralizes up to approximately 1 U/mL of heparin activity.158,159
The majority of clinical laboratories report the PT in seconds along with the INR when monitoring VKA
(see CLSI document H47135).160 The PT-INR is routinely used to monitor VKA (warfarin) therapy and
may also be affected by DTI, direct FXa inhibitors (eg, rivaroxaban)87,161-166 and possibly by indirect FXa
inhibitors (eg, fondaparinux).167 The impact of LA on anticoagulant monitoring is beyond the scope of
this document. For LA testing in general, the PT-INR can be a useful tool in determining sample integrity
or the presence of therapeutic anticoagulant. A prolonged PT suggests an effect of VKA, DTI, direct FXa
inhibitors, or a factor deficiency in the extrinsic or common pathways. Knowledge or suspicion of these
potential interferences will assist with interpretation of LA testing (see Appendix E).
It is rare for LA to prolong standard PT measurements because thromboplastin reagents contain high
concentrations of PL that overcome the in vitro effects of most LA.89,91,110,168 Prolongation of the PT,
when using a recombinant thromboplastin reagent, can be found in a small number of LA patients who
have a very strong LA.168,169 The PT may also be prolonged due to low FII levels (prothrombin) caused by
concomitant non-neutralizing antibodies against prothrombin, resulting in an acquired
hypoprothrombinemia. Unlike most patients with LA, this may cause a bleeding problem.170,171
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8.3 Thrombin Time
The TT, also known as the thrombin clotting time, is a quantitative test to assess the conversion of
fibrinogen to fibrin subsequent to thrombin cleavage of fibrinopeptides A and B from fibrinogen. The TT
is performed as a one-step reaction and represents the time in seconds for fibrinogen to be converted to
fibrin after the addition of a standard concentration of an exogenous source of thrombin (human or
bovine) to citrated plasma (see Appendix D, Figure D11).145 Prolongation of the TT is most commonly
due to:
 Qualitative or quantitative defects in fibrinogen

 Elevated levels of fibrin(ogen) degradation products
 Presence of UFH
 Presence of DTI172
Bovine thrombin is more sensitive to inhibition by the antithrombin/heparin complex than is human
thrombin when tested at low concentrations of UFH.173
In order to ensure an appropriate sample for LA testing (see Appendix A), this guideline recommends
using the TT to determine if pharmacological interferences by UFH or DTI are the cause(s) of a
prolonged APTT. These agents will affect LA assays (see Appendix E) and knowledge of their presence
will assist with LA result interpretation (see Appendixes G1 and G2). Four approaches can be used to
confirm the presence of heparin in a sample:
 Perform TT in the absence and presence of a heparin neutralizing agent.

 Perform both a TT and reptilase time.
 Quantify heparin level by an anti-Xa assay.
 Access the patient’s medical record or contact his/her medical provider.
A prolonged TT is suggestive of heparin, which can be ruled out or confirmed by treating the plasma
sample with a heparin neutralizer (heparinase,137 protamine sulphate, or polybrene174) and repeating the
TT. If the postneutralization plasma TT returns into the RI, then heparin is confirmed. If the
postneutralization TT does not correct into the RI, then the following scenarios are possible:
 The concentration of heparin is greater than the capacity of the neutralizing agent.
 The fibrinogen level is low (hypo- or afibrinogenemia, or the sample is serum).
 A dysfibrinogenemia is present.
 DTI are present.
A Clauss fibrinogen test may be performed to confirm that the fibrinogen level is within its RI and the
sample is suitable for LA testing. Alternatively, both a TT and a reptilase time can be performed.
Reptilase is not affected by heparin. Therefore, a prolonged TT and a normal reptilase time suggest the
presence of heparin (in the absence of a hypo-, dys- or afibrinogenemia). An anti-Xa assay may be
another method in which to quantify the amount of heparin that is present (or absent). If available,
information can be obtained from the patient’s medical record or the nursing station to determine if the
patient had received heparin at or near the time of specimen collection. If there is no alternative but to
perform LA testing on samples that contain heparin above the neutralizing threshold of a particular LA
reagent (as stated in the package insert), then neutralization treatment and subsequent confirmation that
the anticoagulant effect of heparin has been neutralized need to occur before LA testing begins.
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9 Principles of Lupus Anticoagulant Assays

This guideline suggests that APTT, using a reagent responsive to LA (see Section 9.1.1), and dRVVT
(see Section 9.2.1) screening tests are the preferred, first-line LA screening tests. However, this guideline
does not restrict the option to use second-line tests. Assays such as KCT (see Section 9.1.3), TSVT (see
Section 9.2.2), dPT (see Section 9.3.1), etc., detect some LA that do not manifest in APTT and dRVVT
tests, at least a proportion of which are clinically significant.166,175-179 For research purposes, it is
acceptable to perform more than two screening assays.
9.1 Intrinsic Pathway Assays (see Appendix C)
9.1.1 APTT
The APTT is a test using a “partial thromboplastin” because TF is not used to initiate (activate)
coagulation. The APTT is a two-stage assay that consists of activation and recalcification (see Appendix
D, Figure D1). When used as an LA screening test, it is considered an independent test.
The APTT assesses the effect that an LA has on the intrinsic and common pathways of coagulation. It is
one of the oldest tests used to detect LA.25 As noted in Section 8.1, the composition of an APTT reagent
plays a significant role in detecting LA107,180 and this depends primarily on the content of PL.100 The
concept of modifying APTT reagent components to heighten responsiveness to LA has its historical roots
in the dilution of PL used in either PT181 or APTT tests,182 and reagents.183 dAPTT was developed by
Alving et al.183 wherein dilution of PL concentration made it a rate-limiting component in the test
reaction. In the original method, testing was performed on a 1:1 mixture of PPP with NPP using various
dilutions of APTT reagent (ranging from 1:5 to 1:40) and a constant concentration of silica activator.
Subsequently, manufacturers built upon and expanded this concept and created APTT reagents in which
not only PL concentration is lowered but PL type and respective concentrations are varied, as well as PL
configuration. These modifications, in general, yield APTT reagents that show longer clotting times than
those reagents manufactured to be less affected by LA. The contact activator used in the reagent and other
ingredients may also be important,28 although this is disputed.184 Until and if future publications
demonstrate that one activator is superior to others for LA testing, this guideline will not restrict the
choice, thus permitting silica, ellagic acid, and other contact activator-based reagents to be used.184
9.1.2 APTT-based Silica Clotting Time
The SCT assesses the effect of LA on the intrinsic and common pathways of coagulation. The SCT test is
a paired test in that the screening assay, using low levels of PL, is recapitulated in a confirmatory assay
with increased levels of PL, which overwhelm the LA effect (see Appendix D, Figure D4).
The SCT assay was proposed as a substitute for the KCT test because the 2% kaolin suspension proposed
at that time was too turbid and not suitable for analysis in photo-optical devices. The original SCT design
used a colloidal silica preparation that was amenable to automation and gave the same level of sensitivity
as the KCT for detection of LA.185 The colloidal silica preparation remained constant in two reagents with
different levels of PL to make up a screen and a confirmatory reagent where PL concentration (not
composition) was the only difference (10-fold to 100-fold higher in the confirmatory reagent). The
commercially available reagent kit maintains the same concept of consistent silica concentration and low
and high PL levels between screen and confirmatory assays by using an aliquot of the confirmatory PL
reagent to prepare the screen reagent. Hence, the SCT is a silica-based APTT assay similar to the dAPTT
described by Alving.183
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9.1.3 Kaolin Clotting Time
The original KCT was an early screening test for clotting defects.147 The effect of adding kaolin to test
plasmas was to fully activate contact factors and thereby avoid the variable activation that occurred
during sample storage or processing. It was also recognized that platelets shortened the KCT, and because
this was another undesirable variable in test plasmas, a small but consistent quantity of procoagulant PL
was to be added with the kaolin. Ultimately, a higher “optimal” concentration of PL giving the shortest
possible result on normal plasma was used to develop the partial thromboplastin time kaolin (PTTK)
method, which became the prototype APTT.186
The KCT without any added PL was used in early LA studies by Exner et al.187 Because PL seemed to
reduce the effect of LA on most APTT/PTTK tests, the responsiveness to LA was increased when they
were omitted from such reagents. However, the resulting test was very sensitive to endogenous plasma
procoagulant PL mainly released by platelets, which shortened test results. Thus, the KCT uses mixtures
of normal plasma with test plasmas, which also reduces the complicating effects of factor deficiencies and
other abnormalities.187 The KCT is most informative when carried out on fresh PFP or PPP188 from
outpatients rather than frozen PPP that has the potential to contain residual platelet-derived material, or
from patients complicated by clinical problems or receiving treatment. Thus, the KCT RI for frozen PPP
may be wider than that for fresh PPP or PFP.189 The test requires PPP to be preincubated with KCT
reagent to fully activate the contact mechanism. After the addition of calcium chloride, factor XIa
activates the various other clotting factors that interact on the very limited endogenous PL surfaces
available to ultimately form thrombin and a fibrin clot (see Appendix D, Figure D2). LA interfere
strongly with these PL-dependent mechanisms.
For an LDT, the kaolin reagent is easily prepared, inexpensive, and stabile. Commercial KCT reagents
containing low turbidity, nonsettling micronized kaolins, and other silicate minerals for use with
automated analyzers are available. Reports from EQA testing programs, such as ECAT (External quality
Control of diagnostic Assays and Tests, the Netherlands) and RCPA (Royal College of Pathologists of
Australasia) show that the KCT is used by laboratories worldwide.
9.1.4 APTT-based Platelet Neutralization Procedure
The PNP is an APTT-based confirmatory assay for APTT tests used to screen for LA. It was described in
1983 by Triplett et al.,190 and is partly based on observations by Firkin, Howard, and colleagues,191,192 and
others.38,187,193 The PNP is effective in evaluating a prolonged APTT resulting from an LA.9,27,190,194-200 The
PNP involves the addition of washed, freeze-thawed platelets or buffer to patient PPP. The process of
freeze-thawing causes platelet microvesiculation, yielding suspensions enriched in negatively charged
platelet PL. The vesicles can partially absorb LA antibodies that have specificity for PL and associated
proteins, thereby shortening prolonged APTT test results.
APTT testing is performed on two tubes (Tube #1 = patient PPP + buffered saline + APTT reagent, and
Tube #2 = patient PPP + freeze-thawed platelets + APTT reagent) at the same time and clotting times are
compared (see Appendix D, Figure D3). Plasma containing LA will demonstrate significant APTT
shortening (by more than four to five seconds) with addition of freeze-thawed platelets (Tube #2), when
compared to the buffered saline APTT (Tube #1) performed simultaneously. A difference (delta) of less
than four to five seconds between the two tubes relates only to the dilutional effect of the test system.190
The baseline screening APTT must be prolonged in order to perform a PNP. In general, for samples in
which LA is present, the clotting times in seconds for the buffered saline APTT (Tube #1) will be less
prolonged than the baseline screening APTT, provided the same APTT reagent is used. This is because
the inhibitor has been diluted and this is not indicative of a correction.
There are no commercially available PNP reagent kits, though platelet extract material is available from
several vendors. Freeze-thawed platelet extract material can be prepared in-house.190,200,201 Because there
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is a potential for lot-to-lot variability between platelet lysates, which may depend on whether they are
sourced commercially or prepared in-house, laboratories should validate each lot number of platelet lysate
before use. Using in-house preparations (LDTs) may, in some countries, require robust verification and
validation studies before implementation. The APTT reagent used in the test system should be one that is
responsive to the effects of LA (see Section 8.1).
9.1.5 APTT-based Hexagonal Phase Phospholipid Neutralization Test
The HPNT test relies on the principle that LA are neutralized by hexagonal phase egg
phosphatidylethanolamine (HPE) and not with lamellar phase PL.197,202,203 The commercially available
test builds on the principle of the PNP test, but differs from it in that PL is not increased in concentration;
rather, it is in a different configuration. The test also shares similarities with the APTT-based lupus ratio
test204 because both are classified as integrated assays (screening, confirmatory, and mixing test
procedures integrated into one assay), thereby meeting three criteria (see Foreword: Criteria B, C, D) for
the laboratory diagnosis of LA.28 The mixing test component of the assay is met because the screening
and confirmatory procedures are performed on plasma diluted 1:1 with NPP. The APTT reagent used for
integrated tests should be responsive to the effects of LA (see Section 8.1). The commercially available
product contains a heparin neutralizer that neutralizes to approximately 1.0 IU/mL heparin.
APTT testing is performed on two tubes (Tube #1 = patient PPP + buffer + NPP + APTT reagent and Tube
#2 = patient PPP + HPE + NPP + APTT reagent) at the same time and clotting times are compared (see
Appendix D, Figure D5). The addition of HPE to plasma containing LA will result in a shortening of the
APTT (eg, by approximately eight seconds or more) when compared to the prolonged APTT of plasma
with only buffer added.197
9.2 Common Pathway Assays (see Appendix C)
9.2.1 Dilute Russell’s Viper Venom Time
The dRVVT assesses the effect of LA on the common pathway of coagulation. The dRVVT test is
considered a paired test in that the screening assay, using low levels of PL, is recapitulated in a
confirmatory assay with increased levels of PL, which overwhelm the LA effect (see Appendix D, Figure
D6). If the inhibitor is specific for PL/protein complexes, then the extra PL will shorten the confirmatory
dRVVT compared to the screening dRVVT. This occurs because LA antibodies present in patient plasma
will absorb to the extra PL added to the test.
The main procoagulant enzyme in Russell’s viper (Daboia russellii) venom directly activates FX in a
calcium-dependent manner but independent of PL.205 The FXa generated activates prothrombin in the
presence of FVa, Ca2+, and PL. dRVVT reagents from different manufacturers can vary significantly in
LA responsiveness. The same reagents on different instrument types may give different results. Some
reagents use whole Russell’s viper venom, which also contains an FV activator, whereas others may use
only the FX activator.206 The PL used can also vary in type and concentration. Most dRVVT reagents also
contain polybrene to neutralize heparin to approximately 0.8 IU/mL heparin.
The original procedure published by Thiagarajan et al. involved separate additions of dilute PL, Russell’s
viper venom, and calcium to the test plasma.207 By diluting the PL reagent, responsiveness to LA was
increased as shown originally by Exner et al.182 Combining the ingredients with suitable stabilizers
permitted test plasma to simply be mixed with an equal volume of combined reagent and timed to a
clotting end point.208 dRVVT reagents are available commercially from numerous vendors and work well
across most automated coagulometers, except that different RI/cutoff values are needed.69,86
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9.2.2 Other Venom-based Assays
Vipera lebetina venom time: Blunt-nosed viper (V. lebetina) venom, like Russell’s viper venom, will
directly activate FX. A paired test system using V. lebetina venom with low and high PL reagents is
commercially available. This test system is similar in design to the dRVVT test system. However, there
are limited published data to show equivalence and therefore they cannot be considered to be
interchangeable.209
Other Venom Methods (see Appendix D, Figure D7)
Other venom methods are available to screen for LA that, in view of relatively limited data, are best used
in specific clinical circumstances or for research purposes.89,210 These tests would be defined as LDTs and
would require robust verification and validation studies before implementation. Principal among these
tests are two LA screening assays and one confirmatory assay, all of which employ snake venom fractions
containing prothrombin (FII) activators capable of activating des-carboxyprothrombin. As such, they
cannot be affected by VKA, FXa inhibitors, or antibodies against FVIII and FIX.
Textarin time: Textarin is a group D prothrombin activator enzyme complex isolated from the venom
of the Australian Eastern brown snake (Pseudonaja textilis). It activates prothrombin in a PL-, FVa-,
and Ca++-dependent manner. The Textarin time screening assay employs dilution of an appropriate PL
preparation to render the assay responsive to LA, and the Textarin is used at a concentration that
generates clotting times approximating 20 seconds to facilitate reproducibility and permit distinction
between normal plasma and that containing LA.211,212 Textarin was used commercially until recently,
because its availability is limited.
TSVT: The TSVT screening assay employs the prothrombin activator from Coastal Taipan
(Oxyuranus scutellatus) venom, which contains FXa-like and FVa-like subunits and requires PL and
Ca++ as cofactors. Dilution of an appropriate PL preparation renders the assay responsive to LA and
commercially prepared Taipan venom is available at a concentration that generates suitable clotting
times in normal plasma.213 The TSVT is affected by hirudin214 and UFH.212
Ecarin time: Ecarin, derived from the saw scaled viper (Echis carinatus), activates prothrombin in
the absence of PL and FVa.211 Because clot formation occurs in the absence of PL, the Ecarin time is
normal in patients with LA. It is used as a confirmatory assay for both the Textarin time211 and
TSVT.215 Ecarin is affected by DTI when using spiking experiments216-219 and when using
nonspiking experiments.220 Ecarin is not affected by UFH.211
9.3 Extrinsic Pathway Assays (see Appendix C)
9.3.1 Dilute Prothrombin Time
Dilution of a thromboplastin reagent renders the PL concentration rate limiting and allows LA to interfere
with the prothrombinase complex, thereby prolonging clotting times, and is the principle of the dPT as a
screening test for LA. The dPT tests for LA effects on the extrinsic (TF/FVII) and common pathways of
coagulation. The dPT test, as commercially presented, is considered a paired test in that the screening
assay, using low levels of PL, is recapitulated in a confirmatory assay with increased levels of PL, which
overwhelm the LA effect (see Appendix D, Figure D9).
The dPT is conceptually based on the TTI test.181 The dilute “thromboplastin” used in a dPT test is a
recombinant TF, whereas that used in the TTI is of tissue extract. Studies have shown that performance of
the dPT with particular recombinant human TF-containing thromboplastins, at an appropriate high
dilution, is more sensitive to LA than employment of tissue-extract thromboplastin reagents.10,91,190,212,221
The use of the recombinant reagent improved the precision of the method by lowering the clotting times
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and giving better lot-to-lot variability. A confirmatory test can be constructed by using higher
concentrations of the recombinant TF or adding tissue-extract thromboplastin.10,91 A commercial dPT
assay system (screen and confirmatory tests) is available.
9.3.2 Tissue Thromboplastin Inhibition
The TTI test was developed originally by Schleider et al. and has been used for many years.181 The TTI
remains in constant, though limited, use as an LA screening test.10,94,138,176 The source of the tissue-extract
thromboplastin affects assay consistency.
The TTI uses two dilutions of tissue-extract thromboplastin in normal saline (1:50 and 1:500). Patient
plasma is added to these dilutions and then recalcified. NPP is treated in the same way. Patient clotting
times for each dilution are compared to those of NPP run with the same thromboplastin dilutions in order
to generate two normalized ratios (1:50 and 1:500). The final ratio is a screen to confirm ratio: the first
dilution (1:500) operates as a screening test with dilute PL and the other (1:50) as a confirmatory test with
high concentration PL (see Appendix D, Figure D10).
9.4 Overview of Assay Performance
This guideline recommends that clinical laboratories use, whenever possible, commercially available
assays for LA testing. Reagents and reagent kits from manufacturers have undergone robust verification
and validation studies before clinical use. Commercially prepared reagents may also provide limited
variability between reagent lots and batches. For two reasons, this guideline also presents assays as
originally detailed in publications (see Sections 9.1 to 9.3 and Appendix D). First, it is important for the
reader to understand the origins of currently used tests. For example, the TTI, first published in 1976,
showed that:
 Significant dilution of a PT reagent could accentuate (screen) the LA inhibitory effect.

 Decreasing the dilution by 10-fold could attenuate (confirm) the LA effect.
 NPP could be used to normalize test results.
 Screen to confirm normalized ratios could be established.
The second reason for presenting assay principles as first published is to acknowledge that as a global
document, not all laboratories worldwide may have access to manufactured kits or reagents. These latter
laboratories as well as clinical research laboratories, or manufacturers’ research and development
laboratories, will perform these or other assays that are considered “home brew” tests or LDTs.
The popularity of LA tests has changed with time, being influenced by research publications,
convenience, availability, and, most recently, by results from multicenter and international EQA
programs. It should be noted that the latter are usually used for consensus testing only and should not be
considered as method evaluations.
APTT tests are widely used but can vary in their detection of LA. This depends on particular reagent
ingredients including PL (as noted in Section 8.1), which itself does not seem to affect sensitivity to factor
deficiencies or heparin to the same degree as responsiveness to LA. Results from an international EQA
program show that the APTT can be a sensitive screening assay to detect LA provided the reagent is
responsive to LA.222 Differences in LA detection by APTT reagents can be exploited for diagnostic
testing purposes. Publications show that a combination of two matching APTT assays, one responsive to
LA and one not responsive to LA, provides a simple system for detecting LA.150,223 However, a
combination of LA responsive/nonresponsive APTT reagents does not always discriminate for FVIII
inhibitors, whether neutralizing or non-neutralizing.10
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The dRVVT has generally been found to give consistently good results in detecting LA samples in
various EQA challenges.78,94,224 In screening for APS, dRVVT was shown to be more closely linked with
thrombosis than KCT tests because dRVVT seems to detect mostly β2GPI-dependent LA antibodies.22,222
The dRVVT is more specific for LA than APTT, KCT, or dPT tests because it involves only factors (FX,
FV, FII, fibrinogen) of the common pathway. Thus, it is insensitive to factor deficiencies or specific
factor inhibitors acting above FX in the coagulation cascade (such as FVII, FVIII, FIX, FXI, and the
contact factors). Therefore, dRVVT tests are especially useful for discriminating coagulation inhibitors,
detected with APTT tests, as ether possible LA or inhibitors to specific factors (FVIII, FIX, or FXI), the
latter of which are likely to cause bleeding. The dRVVT is not affected in patients where high levels of
FVIII (as in pregnancy) may shorten APTT results and perhaps mask a borderline LA.
The SCT was originally similar to the KCT but later was revised to include a small concentration of PL to
reduce oversensitivity to residual platelets in test samples,185 hence becoming similar to a dilute APTT
test. An SCT/dRVVT combination has been shown to provide a significantly higher association in
identifying LA patients presenting with thrombotic events than other assay combinations.225-227 One study
suggested the improved precision of automated analyzers for the SCT may have contributed to the assay
being more sensitive to patients with low titer LA.228
The KCT, a type of APTT devoid of PL, has at times been considered the most sensitive of all screening
tests for LA, especially when used on dilutions of test plasmas in normal plasma.177,187 It is highly
sensitive and is a good screening test for defects in all clotting factors except FVII. In contrast with the
APTT, the KCT is also responsive to procoagulant PL.229 KCT tests are frequently positive when other
tests are normal.230-232 This may be due to its higher responsiveness to LA or its sensitivity to clotting
factors (and their inhibitors) not detectable with other LA screening tests that may have shorter clotting
time end points. KCT tests are sometimes normal when other LA tests are abnormal. This may be due to a
dilutional effect or because the KCT has a higher sensitivity to antibodies affecting prothrombin rather
than β2GPI-PL complexes.22 However, it can also be due to the presence of activated platelets and
microparticles, which shorten the KCT much more than the dRVVT or other tests.189 Similar to the APTT,
the KCT lacks a complementary confirmatory assay, which may compromise test specificity; therefore,
an independent confirmatory test must be performed.
The dPT screening test using a recombinant human TF–containing thromboplastin plus confirmatory
testing has been shown to detect clinically significant antibodies reactive in dRVVT and APTT
systems.212,222,233 Some clinically significant LA preferentially manifest only in extrinsic pathway-based
assays.91,176,178,234 Therefore, the dPT might be employed as a first-line or second-line test.
Other LA screening and confirmatory tests have been developed based on a variety of coagulation
activators combined with a rate-limiting concentration of PL. A paired test system using V. lebetina
venom rather than Russell’s viper venom is available commercially. Other assays include the TSVT,213
ASLA assay,234 the Textarin time, and the Ecarin time.211 The TSVT and Textarin time tests, which are
insensitive to the effects of VKA, can be useful in patients with a high clinical suspicion of APS who are
receiving VKA and where conventional assays are inconclusive.166,179,211,212,235 However, very low levels
of fibrinogen, low levels of prothrombin, or presence of prothrombin inhibitors may prolong Textarin time
and TSVT results. It is important to recognize that negative results in these tests do not exclude LA
because not all LA are reactive in Textarin time or TSVT. On the other hand, Textarin time and TSVT
have been shown to detect small numbers of LA that do not manifest in conventional assays such as APTT
and dRVVT,179,211,212,235 and they may be useful second-line assays in nonorally anticoagulated patients
with a high clinical suspicion of APS where conventional assays have not revealed an antibody. Some
patients have inhibitors that resist the swamping/neutralizing effect of high PL confirmatory reagents. If
LA is reactive in Textarin time or TSVT, use of the PL-independent Ecarin confirmatory test can reveal
antibodies that may otherwise be uncharacterized.212,236
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Tests that include contact activation (APTT, KCT, and SCT) are sensitive to defects in (and consequently
antibodies against) a wide range of clotting factors, whereas tests using coagulation activators lower in
the clotting mechanism are affected by fewer factors. In theory, the dRVVT, dPT, and TTI tests bypass
contact activation as well as FXI, FIX, and FVIII and should allow good discrimination between LA and
antibodies against these specific factors, which cause bleeding.237 However, in practice this is not entirely
certain. Further, because they involve fewer clotting factors, the TSVT and Textarin tests should be the
most specific tests for PL antibodies while the KCT is probably the least specific for them. The dPT and
ASLA tests may be more specific for PL-dependent antibodies, acting on the TF (extrinsic) pathway and
factors in the common pathway.
10 Assays to Screen for the Presence of Lupus Anticoagulant (Criterion B)
10.1 Available Screening Assays and Their Usage
Current and previous international guidelines recommend the use of at least two screening tests that differ
in assay principles. Examples provided in those guidelines showed, but did not specifically indicate, that
testing should represent two coagulation pathways (intrinsic/common pathway for APTT and common
pathway for dRVVT).27,28 This guideline supports the use of the APTT (using an APTT reagent that is
responsive to LA) and dRVVT as first-line screening tests. Furthermore, this guideline supports limiting
the number of screening tests to two while being cognizant that LA antibody heterogeneity may
necessitate performing additional screening tests (KCT, TSVT, dPT)210 (see Section 13.2). Limiting the
number of screening assays that are used by a laboratory may reduce the incidence of false-positive LA
results (when using a simple statistical model). For example, a laboratory that limits LA screening testing
to two assays and requires both to be abnormal would risk a 1:400 chance that both tests would be falsely
positive (1 false-positive result in 20 with one method + another risk of 1 false-positive result in 20 with
another method = 1:400 chance of false-positive results for both tests). On the other hand, the more
screening assays that are performed and for which abnormality is considered on the basis of a single test,
there is a cumulative risk of false-positive results (four screening tests are performed, three returning
normal results and one returning an abnormal result, in contrast to two screening assays, both returning
normal results). Therefore, the apparent significance of “false-positive” tests is reduced when expressing
them relative to the number of tests performed. For this reason, LA testing cannot be viewed in isolation
and as noted in Section 13, a panel of tests (two screening, confirmatory, and mixing test) should render
the final laboratory diagnosis.
According to recent surveys and questionnaires from EQA testing programs such as ECAT, UK NEQAS
(United Kingdom National External Quality Assessment Service), and RCPA, the APTT and dRVVT, at
the time of this publication, are the most frequently used screening tests for LA (see Table 2). This
conclusion is supported by data from the North American Specialized Coagulation Laboratory
Association.224,238 Surveys from CAP indicate that the APTT is still the most commonly used screening
LA test in the United States.
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Table 2. LA Testing Trends as Observed by EQA Programs

Program ECAT RCPA UK NEQAS
Year 2005 2011 2002 2011 2001 2005 2010
Total 306 448 70 106 231 303 291
Laboratories
Methods
APTT 210 285 61 106 193 268 270
(including SCT)
dRVVT 199 289 70 100 225 290 271
KCT 39 20 43 29 78 82 36
dPT (TTI) 24 23 0 1 15 23 22
Others* 5 0 0 0 28 54 11
*
Others include TSVT, Textarin time, and the ASLA assay.
Abbreviations: APTT, activated partial thromboplastin time; ASLA, activated seven lupus anticoagulant; dPT, dilute prothrombin
time; dRVVT, dilute Russell’s viper venom time; ECAT, External quality Control of diagnostic Assays and Tests; EQA, external
quality assessment; KCT, kaolin clotting time; LA, lupus anticoagulant(s); RCPA, Royal College of Pathologists of Australasia;
SCT, silica clotting time; TSVT, Taipan snake venom time; TTI, tissue thromboplastin inhibition; UK NEQAS, United Kingdom
National External Quality Assessment Service.
This guideline does not recommend quantifying or semiquantifying LA. There are no publications that
provide guidance to a laboratory for titering LA with clot-based assays. When attempts have been made
to titer LA, potency has not been shown to correlate well with thrombotic risk.239 There also has not been
a thorough evaluation of which LA screening tests may be better for establishing the diagnosis of APS.
10.2 APTT
When a laboratory needs to determine the absence or presence of LA, then an APTT reagent must be used
that demonstrates responsiveness to LA (see Section 8.1) either by in-house validation (see Section 6.2)
or documentation from a manufacturer. One cannot quantify the detection of LA by an APTT reagent.
The APTT is not routinely directly paired to a matching confirmatory assay and is therefore considered an
independent LA screening test. Confirmatory assays that can be used in conjunction with an APTT
screening test include the PNP described in Section 11.1 and the HPNT described in Section 11.2.
Alternatively, a laboratory could pair two different APTT reagents, one responsive to LA and the other
one high in PL content so that it is not affected by LA.223 A caveat is that any APTT reagent can be
responsive to LA if the antibody is sufficiently potent.
10.2.1 Calculation of Test Results
APTT results can be expressed as clotting times in seconds (s). This guideline recommends that APTT
screening test results be calculated and reported as a normalized ratio (see Appendix F). Patient APTT
clotting time in seconds is divided by the clotting time in seconds of the mean of the RI.
Normalized Screen Ratio = APTT Screen Patient (s) / APTT Screen Mean of RI (s)
NOTE: Normalization to mean of RI is recommended in this guideline; however, manufacturers’

instructions must be followed if they differ from this recommendation.
10.2.2 Limitations of APTT (see Appendix E)
 If possible, samples from patients treated with UFH should not be screened with the APTT for the
presence of LA because correct interpretation of test results is difficult. 137 However, plasma samples
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may be treated with a heparin neutralizer (heparinase, protamine sulphate, or polybrene) and
subsequent confirmation that the anticoagulant effect of heparin has been neutralized needs to occur
(see Section 8.3) before undertaking APTT testing. LMWH, depending upon their composition and
circulating concentration, may prolong the APTT and therefore results should be interpreted with
caution.240 However, in certain patient populations that are at high risk for APS and treated with
LMWH, there is no alternative but to test in the presence of the drug.
 APTT screening test is affected by VKA, and, if possible, samples should not be screened with the
APTT for the presence of LA because correct interpretation of test results is difficult.241
 APTT screening assay is affected by DTI or FXa inhibitors, thereby making correct interpretation of
test results difficult.87,161,164-167,242,243
 Plasmas from patients with hereditary or acquired deficiencies of FXI, FIX, FVIII, FX, FV, FII, and
fibrinogen as well as contact factors (FXII, prekallikrein [PK], or high molecular weight kininogen
[HMWK]) may give a prolonged APTT test result.
 FVIII, FIX, and FXI antibodies from hemophilia A, B, and C patients, respectively, as well as
acquired inhibitors to these factors affect the APTT.
 Increased levels of FVIII, as seen, for example, in the third trimester of pregnancy, could mask a
borderline LA121 or attenuate the in vitro anticoagulant effect of another, making it appear less potent.
 The presence of residual platelets in the test plasma can strongly influence results, especially if these
platelets are activated by freeze-thawing. 188,244 The residual platelet count must be < 10 × 109/L (see
Section 7.3.1).
10.3 APTT-based Silica Clotting Time
The SCT screening test is a silica-based APTT assay using low levels of PL. A reagent kit is available
commercially.
SCT screening results can be expressed as clotting times in seconds (s). This guideline recommends that
SCT screening test results be calculated and reported as a normalized ratio (see Appendix F). Patient SCT
screening clotting time in seconds is divided by the clotting time in seconds of the mean of the RI for the
SCT screening test.
Normalized Screen Ratio = SCT Screen Patient (s) / SCT Screen Mean of RI (s)
10.3.2 Limitations of Silica Clotting Time Screening Assay (see Appendix E)
 If possible, samples from patients treated with UFH should not be screened with the SCT for the
presence of LA because correct interpretation of test results is difficult.185 The commercially available
SCT screening reagent contains a heparin neutralizer (level noted in product insert) that permits
testing. However, plasma samples containing UFH levels greater than the stated level may give
incorrect results. Plasma samples may be treated with a heparin neutralizer (heparinase, protamine
sulphate, or polybrene) and subsequent confirmation that the anticoagulant effect of heparin has been
neutralized needs to occur (see Section 8.3) before undertaking SCT screening testing. LMWH,
depending upon their composition and circulating concentration, may prolong the SCT screening test;
therefore, results should be interpreted with caution.225 However, in certain patient populations that
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are at high risk for APS and treated with LMWH, there is no alternative but to test in the presence of
the drug.
 SCT screening assay is affected by VKA and liver disease.185
 SCT screening test is usually not affected by the presence of fondaparinux; however, this may be
reagent dependent.245
 Plasmas from patients with hereditary or acquired deficiencies of FXI, FIX, FVIII, FX, FV, FII, and
fibrinogen as well as contact factors (FXII, PK, or HMWK) may give a prolonged SCT test result.
 FVIII, FIX, and FXI antibodies from hemophilia A, B, and C patients, respectively, as well as
acquired inhibitors to these factors affect the SCT screening test.
 The presence of residual platelets in the test plasma can strongly influence results, especially if these
platelets are activated by freeze-thawing.246 The residual platelet count must be < 10 × 109/L (see
Section 7.3.1). Platelets in freeze-thawed plasmas shorten SCT results by releasing procoagulant PL
into the plasma.246
10.4 Dilute Russell’s Viper Venom Time
A procoagulant enzyme in Russell’s (D. russellii) viper venom directly activates FX in a calcium-
dependent manner but independent of PL. The dRVVT screening test uses low levels of PL. Reagents are
available commercially from various manufacturers.
Results for dRVVT screening tests can be expressed as clotting times in seconds (s). This guideline
recommends that dRVVT screening test results be calculated and reported as a normalized ratio (see
Appendix F). Patient dRVVT screening clotting time in seconds is divided by the clotting time in seconds
of the mean of the RI for the dRVVT screening test.
Normalized Screen Ratio = dRVVT Screen Patient (s) / dRVVT Screen Mean of RI (s)

10.4.2 Limitations of Dilute Russell’s Viper Venom Time Screening Assay (see Appendix E)
 Most commercially available dRVVT screening reagents contain a heparin neutralizer (level noted in
product insert) that permits testing in the presence of UFH. However, plasma samples containing
UFH levels greater than the denoted level may give incorrect results. Plasma samples may be treated
with a heparin neutralizer (heparinase, protamine sulphate, or polybrene) and subsequent
confirmation that the anticoagulant effect of heparin has been neutralized needs to occur (see Section
8.3) before undertaking dRVVT screening testing.
 Most patients on VKA (acquired deficiencies of FVII, FX, and FII) have prolonged dRVVT clotting
times.89,247
 DTI (argatroban, bivalirudin, dabigatran, and hirudin) and FXa inhibitors (fondaparinux and
rivaroxaban) give prolonged dRVVT results that show only partial correction in a dRVVT screening
mixing test. Hirudin,87,243 bivalirudin,243 and dabigatran248 cause elevated dRVVT results. Argatroban
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prolongs the dRVVT screen.87,243 Fondaparinux did not affect the dRVVT in one study.243
Rivaroxaban causes prolongation of the dRVVT screening assay.166 DTI prolong the TT, which is not
prolonged with a typical LA. Chromogenic anti-Xa assays can be used to discriminate FXa inhibitors
from LA. Unless the reagent manufacturer provides evidence that DTI and FXa inhibitors do not
interfere with their dRVVT assays, testing should only be performed in the absence of these agents.
 Plasmas from patients with hereditary or acquired FII, FV, and FX deficiencies give a prolonged
dRVVT test result. FV inhibitors give prolonged dRVVT results that do not correct with NPP. These
patients have typically a very prolonged PT, low levels of FV, and associated bleeding. Some
inhibitors to FV are directed against its PL-binding domain and masquerade as LA.154,249,250
 Residual platelets in PPP, due to insufficient centrifugation, can give false-negative results if such
plasmas have been frozen and then thawed. The residual platelet count must be < 10 × 109/L (see
Section 7.3.1).
 Reagents for dRVVT testing vary across different suppliers and this may affect results with different
interfering substances. Therefore, it is important for publications to specify which reagent has been
used when assessing interference by therapeutic substances.
10.5 Dilute Prothrombin Time
The dPT uses a recombinant TF in the presence of diluted PL to screen for LA. A reagent kit is available
commercially.
Results for dPT screening tests can be expressed as clotting times in seconds (s). This guideline
recommends that dPT screening test results be calculated and reported as a normalized ratio (see
Appendix F). Patient dPT screening clotting time in seconds is divided by the clotting time in seconds of
the mean of the RI for the dPT screening test.91
Normalized Screen Ratio = dPT Screen Patient (s) / dPT Screen Mean of RI (s)
10.5.2 Limitations of Dilute Prothrombin Time Screening Assay (see Appendix E)
 To varying degrees, prolonged test results are noted with UFH, VKA, DTI, or FXa inhibitors. The
commercially available dPT screening reagent contains a heparin neutralizer (level noted in product
insert) that permits testing in the presence of UFH. However, plasma samples containing UFH levels
greater than the denoted level may give incorrect results. Plasma samples may be treated with a
heparin neutralizer (heparinase, protamine sulphate, or polybrene) and subsequent confirmation that
the anticoagulant effect of heparin has been neutralized needs to occur (see Section 8.3) before
undertaking dPT screening testing.
 To varying degrees, prolonged test results may be seen with deficiencies of FII, FV, FVII, or FX;
specific inhibitors of FII, FV, FVII, or FX; or a marked reduction in fibrinogen level. Severe
deficiencies of FVIII and FIX can prolong dPT results.91,190 Dilution of thromboplastin (TF activator)
increases the sensitivity of the test to factor deficiencies in both the extrinsic pathway (direct
activation of FX by FVIIa/TF) and intrinsic pathway (indirect activation of FX when FVIIa/TF
activates FIX).91,190
 For LDT assays, thromboplastin reagent heterogeneity is a concern because sensitivity and specificity
not only varies between reagents (between species or between manufacturers) but also between
batches of the same reagent.
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 For LDT assays, various approaches have been adopted for thromboplastin dilutions, resulting in
significant differences in diagnostic performance.94 Although sensitivity is improved when employing
a diluted recombinant human TF thromboplastin, an optimal dilution cannot be suggested that is
applicable to every analytical platform.
10.6 Kaolin Clotting Time
The KCT is an APTT-based LA screening test in which no exogenous PL is added to the test. Reagent
kits are available commercially. The KCT is affected by many clotting abnormalities. Therefore, it can be
made more specific by diluting patient plasma with NPP instead of testing neat plasma.
Recommendations by commercial suppliers are that plasma be diluted 1:1 with NPP (analogous to a
traditional mixing test) as well as 1:4 (one part patient PPP to four parts NPP).
This guideline recommends that KCT screening test results should be calculated and reported as a
normalized ratio (1:4 mixing test) or as an ICA for the 1:1 mixing test (see Appendix F).
KCT normalized ratio (delta KCT) using a 1:4 mixing test (s = seconds):
delta KCT = [KCT Screen 1:4 Mix (s) − KCT Screen NPP (s)] / KCT NPP (s)
NOTE: Because the KCT is performed on diluted plasma, normalization to NPP and not to the mean of
the RI is suggested by most manufacturers’ product inserts.
The ICA (Rosner Index) was originally designed to be used with the KCT.62
[KCT Screen 1:1 Mix (s) − KCT Screen NPP (s) / KCT Screen Patient (s)] • 100
10.6.2 Limitations of Kaolin Clotting Time (see Appendix E)
 UFH affects the KCT and this may persist in a 1:1 mixture with NPP. If possible, samples from
patients treated with UFH should not be screened with the KCT. However, plasma samples may be
treated with a heparin neutralizer (heparinase, protamine sulphate, or polybrene) and subsequent
8.3) before undertaking KCT testing.
 VKA will prolong KCT tests but these effects are reduced because testing is performed using plasma
that is diluted 1:1 with NPP. Using 1:4 dilutions may further mitigate the VKA effect as more NPP is
available to correct the factor deficiencies caused by VKA therapy.
 Therapeutic anticoagulants such as DTI and FXa inhibitors will prolong KCT tests and these effects
may not be reduced by using 1:1 dilutions with NPP. Using 1:4 dilutions also may not return results
to normality even though the inhibitors are further diluted by NPP.
 The KCT may be prolonged with factor deficiencies (all but FVII) and with inhibitors to specific
factors. In general, the KCT uses dilutions of patient plasma with NPP to mitigate these effects.
 Because no PL is added in this test, the clotting time result is strongly dependent on the endogenous
content of PL in a plasma sample as well as on the activity of most clotting factors. Thus, residual
platelets in freeze-thawed plasmas can contribute to a short result, possibly bypassing weaker titer
LA. Plasmas to be tested with a KCT should be fresh and double-centrifuged. Though filtration of
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plasma before freezing also has been suggested for LDT assays,188 this guideline does not recommend
filtration of PPP for clinical testing.
11 Assays to Confirm the Presence of Lupus Anticoagulant (Criterion C)

A confirmatory test is essential for identifying LA.27 These antibodies differ from other coagulation
inhibitors because the presence of an excess of anionic PL reduces or neutralizes their effect on
coagulation. Correction of a prolonged clotting time by the addition of extra PL confirms the presence of
LA. Confirmatory assays are performed using the same assay type that resulted in a positive screening
test. If an independent, intrinsic pathway screening test such as the APTT was prolonged, then, for
example, a PNP confirmatory assay could be used. If a paired, common pathway screening assay such as
the dRVVT was prolonged, then a dRVVT confirmatory test is used. If both screening assays are
prolonged, then confirmatory assays should be performed on both tests because multiple positivity is
prognostically significant.251 For paired tests (SCT, dRVVT, and dPT), the confirmatory test result should
not be viewed in isolation because certain interferences to the confirmatory test itself (see Appendix E)
may initially lead to spurious results. However, when this result is used with its paired screening test
result to calculate a normalized ratio, normalization compensates for prolongations in both screening and
confirmatory assays and therefore may not yield a false-positive paired test interpretation.
There are four ways to confirm the presence of an LA:
1. When using paired test systems, confirmatory assays should be used in conjunction with the
accompanying paired screening test. Examples are assays based on the intrinsic pathway (SCT),
extrinsic pathway (dPT), or the common pathway (dRVVT). Confirmatory and screening assay
designs are similar within a paired test system with only the levels of PL differing, ie, high
concentrations in confirmatory assays in contrast to low concentrations in screening assays. Screening
and confirmatory tests should be performed ideally on the same aliquot from the same freeze/thaw
cycle, within the four-hour stability period.
2. A number of screening tests, including the APTT, are not available in combination with or directly
paired to a confirmation test. To confirm the presence of LA, these independent screening assays
should be combined with an independent confirmatory assay with high levels of PL. Some APTT
reagents are PL rich and can be used in conjunction with an APTT screening reagent that is PL
“poor.” Alternatively, PL can be sourced from either washed platelets or freeze-thawed platelets or be
prepared/presented in a different configuration.194,198 The PNP is an APTT-based confirmatory assay
that uses PL derived from platelets. Phosphatidylserine containing liposomes and cardiolipin have
been considered as alternative PL sources in clot-based assays, but, to date, these have not shown to
be sufficiently robust to be used in a diagnostic setting.18
3. A third confirmatory choice is the use of an integrated assay (HPNT) that incorporates three criteria
(screening, confirmation, and mixing) for LA testing into a single assay.202 PL in this assay are in a
hexagonal (II) phase configuration.202
4. A fourth possibility is the use of the Ecarin clotting time as a confirmatory assay. The Ecarin clotting
time in combination with the Textarin clotting time (see Section 9.2.2) has been used, but Textarin
availability is limited. The Textarin/Ecarin ratio is a test based on the differential dependence of
snake venom on PL to activate the coagulation pathway.211 Ecarin activates prothrombin
independently of PL. The Ecarin clotting time can also be used as a confirmatory test for the TSVT
screening assay.215
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11.1 APTT-based Platelet Neutralization Procedure
The PNP is an APTT-based LA confirmatory test that uses suspensions enriched in negatively charged
platelet PL to dampen or neutralize the inhibitory effect of LA antibodies. These suspensions are available
commercially.
PNP results are expressed as delta clotting times in seconds (s) between two tubes (Tube #1 = patient PPP
+ buffered saline + APTT and Tube #2 = patient PPP + freeze-thawed platelets + APTT), which are
performed at the same time. A delta value can be determined by subtracting Tube #2 values from Tube #1
values.
Delta (s) = Tube #1 (PPP + buffered saline) − Tube #2 (PPP + freeze-thawed platelets)
It is important to note that the buffered saline APTT derived from Tube #1 must be prolonged in order to
obtain reliable results with this assay. Furthermore, the baseline, preliminary APTT screening test must
also be prolonged.
11.1.2 Limitations of Platelet Neutralization Procedure (see Appendix E)
 The presence of UFH will usually give false-positive results (abnormal delta)190 because the PNP
reagent is enriched in platelet factor 4 (PF4), a heparin-neutralizing protein. The distinction is usually
not difficult, because heparin substantially prolongs the TT.173 Plasma samples may be treated with a
heparin neutralizer (heparinase, protamine sulphate, or polybrene) and subsequent confirmation that
the anticoagulant effect of heparin has been neutralized needs to occur (see Section 8.3) before
undertaking PNP testing.
 The presence of coagulation FV inhibitors or FV deficiency may also produce false-positive results
(abnormal delta), reflecting the addition of FV in the PNP reagent (platelets contain about 20% to
25% of FV in blood).252,253 In such cases, the PT typically is significantly prolonged, and coagulation
factor assays can confirm isolated decreased FV activity. The PNP is usually able to differentiate
deficiencies or inhibitors of specific coagulation factors (eg, FVIII) from LA.190,194
 In-house-prepared freeze-thawed platelets can be used; however, preparations can show batch-to-
batch inconsistency, probably due to the presence of variable amounts of PF4254 and FV.
11.2 APTT-based Hexagonal Phase Phospholipid Neutralization Test
The HPNT is APTT based and incorporates a screening, confirmatory, and mixing step into one assay
system. A kit is available commercially.
HPNT results are expressed as delta clotting times in seconds (s) between two tubes (Tube #1 = patient
PPP + buffer + NPP + APTT reagent and Tube #2 = patient PPP + HPE + NPP + APTT reagent), which are
performed at the same time. A delta value can be determined by subtracting Tube #2 values from Tube #1
values.
Delta (s) = Tube #1 (PPP + buffer + NPP) − Tube #2 (PPP + HPE + NPP)
NOTE: The manufacturer’s claims for the commercial product state that this is a qualitative assay that
can be reported as positive (LA present) or negative (LA not detected).
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11.2.2 Limitations of Hexagonal Phase Phospholipid Neutralization Test (see Appendix E)
 The commercially available HPNT kit contains a heparin neutralizer (level noted in product insert)
that permits testing in the presence of UFH.197 However, plasma samples containing UFH levels
greater than the denoted level may give incorrect results (abnormal delta). Plasma samples may be
treated with a heparin neutralizer (heparinase, protamine sulphate, or polybrene) and subsequent
8.3) before undertaking HPNT testing.
 Some LMWH preparations may not affect the HPNT.255
 VKA did not cause false-positive results (abnormal delta) in the HPNT when testing specimens with
INR 1.5 to 4.3.197
 DTI may give false-positive results (abnormal delta) and specifically argatroban (Van Cott E,
unpublished observations, 2010) and dabigatran have been shown to cause false-positive HPNT
results.
 Specific factor inhibitors, particularly to FVIII and FV, may give false-positive results.154,196
11.3 APTT-based Silica Clotting Time Confirmatory Test
The APTT-based SCT is a paired test system in which the screening assay is recapitulated with the use of
high levels of PL. A paired test system is commercially available.
SCT confirmatory results can be expressed as clotting times in seconds (s). This guideline recommends
that SCT confirmatory test results be calculated and reported as a normalized ratio (see Appendix F).
Patient SCT confirmatory clotting time in seconds is divided by the clotting time in seconds of the mean
of the RI for the SCT confirmatory test.
Normalized Confirmatory Ratio = SCT Confirm Patient (s) / SCT Confirm Mean of RI (s)
For paired test calculation:
Results from both the screening and confirmatory assays are expressed as a ratio between these two tests.
This guideline recommends that the screening to confirmatory ratio be expressed as a normalized ratio.
The screen normalized ratio (patient SCT screen clotting time in seconds divided by the mean of the
screen RI in seconds) is divided by the confirmatory normalized ratio (patient SCT confirmatory clotting
time in seconds divided by the mean of the confirmatory RI in seconds) (see Appendix F).
Normalized Screen to Confirmatory Ratio = [Screen Patient (s) / Screen Mean of the RI (s)] / [Confirm
Patient (s) / Confirm Mean of the RI (s)]
11.3.2 Limitations of Confirmatory Silica Clotting Time (see Appendix E)
 Using only the confirmation procedure does not discriminate between LA and the presence of UFH.
The commercially available SCT confirmatory reagent contains a heparin neutralizer (level noted in
UFH levels greater than the denoted level may give prolonged results. Plasma samples may be treated
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8.3) before undertaking SCT confirmatory testing.
 SCT confirmatory clotting times are prolonged by VKA, factor deficiencies of the intrinsic pathway
(FVIII, FIX, FXI, and FXII), and specific factor inhibitors.
 DTI or FXa inhibitors can affect test results.248
NOTE: Normalization of screening to confirmatory test results compensates for prolongations in both
screening and confirmatory assays and therefore may not yield a false-positive paired test interpretation.
11.4 Dilute Russell’s Viper Venom Time Confirmatory Test
The dRVVT is a paired test system in which the screening assay is recapitulated with the use of high
levels of PL. This paired test system is available commercially from numerous vendors.
dRVVT confirmatory results can be expressed as clotting times in seconds (s). This guideline
recommends that dRVVT confirmatory test results be calculated and reported as a normalized ratio (see
Appendix F). Patient dRVVT confirmatory clotting time in seconds is divided by the clotting time in
seconds of the mean of the RI for the dRVVT confirmatory test.
Normalized Confirmatory Ratio = dRVVT Confirm Patient (s) / dRVVT Confirm Mean of RI (s)
This guideline does not support a dRVVT screening to dRVVT confirmatory ratio in the absence of
normalization because this simple ratio does not take into account differences in operator and/or analyzer
performance or reagent quality and stability issues. Therefore, this guideline recommends that a ratio of
dRVVT screening and confirmatory test results be calculated and reported as a normalized ratio. The
screen normalized ratio (patient dRVVT screening clotting time in seconds divided by the screening
clotting time in seconds of the mean of the RI) is divided by the confirmatory normalized ratio (patient
dRVVT confirmatory clotting time in seconds divided by the confirmatory clotting time in seconds of the
mean of the RI) (see Appendix F).
Normalized Screen to Confirmatory Ratio = [Screen Patient (s) / Screen Mean of RI (s)] / [Confirm
Patient (s) / Confirm Mean of RI (s)]
Alternatively, a percent correction of normalized ratios can be used. The confirm normalized ratio (patient
dRVVT confirmatory clotting time in seconds is divided by the confirmatory clotting time in seconds of
the mean of the RI) is subtracted from the screen normalized ratio (patient dRVVT screening clotting time
in seconds divided by the screening clotting time in seconds of the mean of the RI) with all divided by the
screen normalized ratio (patient dRVVT screening clotting time in seconds divided by the screening
clotting time in seconds of the mean of the RI). The entire value is then multiplied by 100 (see Appendix
F).
% Correction of Normalized Ratios = [(Screen Patient / Screen RI Mean) − (Confirm Patient / Confirm
RI Mean) / (Screen Patient / Screen RI Mean)] • 100

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11.4.2 Limitations of Confirmatory Dilute Russell’s Viper Venom Time (see Appendix E)
 The dRVVT confirmatory test does not always discriminate between LA and the presence of UFH.
Most commercially available dRVVT confirmatory reagents contain a heparin neutralizer (level noted
in product insert) that permits testing in the presence of UFH. However, plasma samples containing
UFH levels greater than the denoted level may give prolonged results. Plasma samples may be treated
8.3) before undertaking dRVVT confirmatory testing.
 VKA may yield false-positive test results.256,257
 DTI may cause false-positive dRVVT confirmatory test results. Argatroban spiked into LA-positive
samples caused false-negative dRVVT normalized screen to confirm ratios in some samples.87 In
another study, two other DTI (bivalirudin and hirudin) caused false-positive dRVVT confirmatory
ratios whereas argatroban did not.243
 Rivaroxaban, a direct FXa inhibitor, caused dRVVT normalized screen to confirm ratios to be falsely
positive or indeterminate in a recent study. However, the dRVVT test system was not specified and
findings may not apply to all dRVVT reagents.258
 Some FV inhibitors are directed against the PL binding site of FV and can masquerade as LA. These
FV inhibitors show correction with the addition of PL, thereby making it difficult to discriminate
between them and LA.99,154,249
 Confirmatory reagents can also alter the dRVVT results of plasmas where LA is not detected. The
effect of the confirmation reagents on normal plasmas depends on the commercial supplier of the
reagents. The sensitivity and specificity of confirmatory tests not only depend on the commercial
assay but are also influenced by the type of coagulometer.69
11.5 Dilute Prothrombin Time Confirmatory Test
The dPT is a paired test system in which the screening assay is recapitulated with the use of high levels of
PL. A paired test system is available commercially or can be devised as an LDT.
Confirmatory results for dPT can be expressed as clotting times in seconds (s). This guideline
recommends that dPT confirmatory test results be calculated and reported as a normalized ratio (see
Appendix F). Patient dPT confirmatory clotting time in seconds is divided by the clotting time in seconds
of the mean of the RI for the dPT confirmatory test.
Normalized Confirmatory Ratio = dPT Confirm Patient (s) / dPT Confirm Mean of RI (s)
This guideline recommends that a ratio of dPT screening and confirmatory test results be expressed as a
normalized ratio. The screen normalized ratio (patient dPT screen clotting time in seconds divided by the
screening clotting time in seconds of the mean of the RI) is divided by the confirmatory normalized ratio
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(patient dPT confirmatory clotting time in seconds divided by the confirmatory clotting time in seconds of
the mean of the RI).
Normalized Screen to Confirmatory Ratio = [Screen Patient (s) / Screen Mean of RI (s)] / [Confirm
Patient (s) / Confirm Mean of RI (s)]
11.5.2 Limitations of Confirmatory Dilute Prothrombin Time (see Appendix E)
 The commercially available dPT confirmatory reagent contains a heparin neutralizer (level noted in
UFH levels greater than the denoted level may give incorrect results. Plasma samples may be treated
8.3) before undertaking dPT confirmatory testing.
 VKA may give prolonged clotting times
 Some FV inhibitors show correction with the addition of PL, thereby potentially giving false-positive
test results.
12 Mixing Test as Applied to Screening, Confirmatory, and Integrated Assays

(Criterion D)
This guideline, recognizing the limitations of a mixing test (see Section 12.4), has reprioritized the
importance of it in LA testing. As shown in Appendix A, this guideline supports the initial performance
of LA screening and confirmatory assays to show the PL dependence of the antibody. If PL dependence
cannot be demonstrated at the LA confirmatory assay step (see Figures A1 and A2 in Appendix A), then a
mixing test is performed to assess the inhibitory action of LA in patient plasma against NPP. Therefore,
this guideline does not dismiss the use of a mixing test and continues to support its inclusion, when
necessary, as part of a comprehensive LA testing panel because it can increase diagnostic specificity.
However, a mixing test does not add to diagnostic specificity when testing algorithms show PL
dependence (LA present) and there is no evidence for other causes of elevated clotting times.
Essentially, a mixing test uses NPP that interacts with patient-citrated PPP to either correct a coagulation
factor deficiency in the patient’s plasma or the NPP is adversely affected (clotting time prolongs) by an
abnormality (inhibitor) in the patient’s plasma. This applies to preliminary tests (PT, APTT), either
screening (APTT, dRVVT, SCT, KCT, and dPT) or confirmatory (dRVVT, SCT, and dPT) LA assays, or
an integrated LA test system (HPNT). A mixing test consists of two reagent components: test system
reagent (eg, PT, APTT, dRVVT) and NPP. Reagent responsiveness to LA varies widely, as noted in
Sections 8.1 and 10.2. Consequently, a mixing test is only as good as the “parent” test on which it is
based.
A mixing test mixes together patient PPP and NPP, the outcome of which is the dilution of components in
each of the respective plasmas. The mixing test most widely used adds equal amounts (1:1 or 50% to
50%) of patient PPP and NPP (see Appendix D, Figure D12a) to a tube, which is then tested in the same
assay system as the original (prolonged) sample. The underlying premise is that 50% of any factor is
sufficient to yield a normal clotting time. Therefore, a 50% contribution of factors by NPP should
“correct” factor(s) deficiency present in patient PPP. Failure to “correct” is attributed to the activity of an
inhibitor in patient PPP directed against NPP. It is common for the abnormality to appear less marked in
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the mixing test due to the dilution effect. The presence of 50% NPP in the assay may overwhelm the
effects of a low titer inhibitor in patient PPP.10,29,169,179,257
12.1 Reasons for Performing a Mixing Test
12.1.1 Mixing Test and Routine Prothrombin Time and APTT
The mixing test is used in routine coagulation practice following a prolonged routine screening test (PT
and APTT). It is important to note that a mixing test yields no valuable information if the initial clotting
time is not prolonged. A 1:1 mixing test with a preliminary APTT (see Section 8.1) may be informative
because the classical presentation of LA is that the mixing test does not “correct” due to the inhibitory
nature of LA. However, the responsiveness of the APTT reagent to LA, dilution effect, and suitability of
NPP can cause the mixing test to “correct” and suggest that an inhibitor is not present. Thus, LA analysis
should still be undertaken because correction of a preliminary APTT mixing test does not exclude the
presence of LA, particularly one with low titer.
For samples with prolonged clotting times, a mixing test directs subsequent evaluation to either factor(s)
deficiency or an inhibitor. A 1:1 mixture of PPP and NPP that is tested immediately upon mixing is
referred to as an immediate mixing test. Criteria for “correction” or “no correction” are determined and
validated by a laboratory subsequent to establishing RI (see Section 6.3 and Appendix B) specific for
each mixing test assay (eg, APTT mixing test, dRVVT screening mixing test). (See CLSI document
H47135 for information on correction criteria related to PT and APTT testing.) If a clotting time for a PT
or APTT immediate mixing test shows “correction” (ie, return to an RI), then the probability is high that a
factor deficiency is present, though its cause (congenital or acquired) would not be discernible.
In contrast, a failure to correct (ie, results do not return to an RI) would be indicative of an inhibitor;
however, the type (eg, LA or factor-specific inhibitor) cannot be distinguished. Inhibitors that exert an
effect on normal plasma have historically been termed circulating anticoagulants. They encompass four
broad categories:
 Nonspecific antibodies such as LA

 Specific antibodies against specific coagulation factors (eg, FVIII and FV)
 Nonspecific inhibitors such as UFH, indirect FXa inhibitors, paraproteins, and fibrin split products
 Specific inhibitors such as DTI and direct FXa inhibitors
Some FVIII inhibitors may show a correction with an APTT mixing test because these antibodies are time
dependent, ie, they do not react immediately but require time for their neutralizing effect to be
demonstrated. While LA and specific factor inhibitors more commonly affect the APTT, one cannot
dismiss that when potent, the LA can prolong the PT and that rare inhibitors to factors in the common
pathway (assessed by the PT) may also occur.
12.1.2 Mixing Test and Lupus Anticoagulant Screening Assays
This guideline recommends that a mixing test for screening assays be performed subsequent to finding
both screening and confirmatory LA test results to be abnormal (see Appendix A, Figures A1 and A2). In
most cases, LA is expected to produce a “no correction” or “partial correction” result with a 1:1
immediate screening mixing test. If clotting times “correct,” there is a high degree of suspicion for a
factor deficiency (congenital or acquired, such as VKA), thereby increasing test specificity. However,
correction of a screening mixing test does not exclude the presence of LA.28,29,169,257,259-263
A mixing test may be performed with any of the dilute PL reagents used in LA screening tests (ie, APTT,
dRVVT, SCT, dPT, KCT). Often this is an advantage given that the degree of clotting time prolongation
of a patient sample is typically greater with a dilute PL reagent. This is particularly so when comparing
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APTT reagents that have been modified to detect LA in contrast to those that have not been so treated.
The greater prolongation may make it easier to interpret a 1:1 mixing test. A mixing test must be
performed with the same assay that showed the prolongation (ie, dRVVT screening mixing test is
performed because dRVVT screening assay is prolonged).
It has been suggested that up to 15% of LA are time dependent; specifically, an inhibitor effect is not seen
unless the sample is incubated at 37°C for a minimum of one hour.264 However, this time dependency of
LA is often reagent dependent and related to pH increases during incubation.189 Therefore, more strongly
buffered APTT reagents, which better resist pH changes, show less prolongation in clotting times over
time. Changes in pH can also be prevented by capping sample tubes. For the above noted reason,
incubated mixing tests are not recommended for LA testing.
12.1.2.1 Lupus Anticoagulant Cofactor Effect
The LA cofactor effect is characterized by a paradoxical further prolongation of a patient’s clotting time
upon mixing with NPP, and while suggestive of LA, it is not pathognomonic.265 Though not common, the
phenomenon is most frequently detected by the APTT. This observation is reagent dependent and affected
by PL concentration, presence of a very strong LA, and the proportion of LA plasma and NPP, all
suggesting a prozone effect.169,266 It is referred to as the cofactor effect because a patient’s plasma may be
deficient in a plasma cofactor essential for LA to exert its in vitro anticoagulant effect. Mixing with NPP
supplies the cofactor, which has been proposed to be prothrombin or β2GPI.267,268
12.1.3 Mixing Test and Lupus Anticoagulant Confirmatory Assays
Mixing tests are usually performed with LA screening assays. However, a mixing test with an LA
confirmatory assay (see Appendix A, Figure A2) may be performed if the confirmatory test on undiluted
plasma is prolonged (above an established cutoff value). It is important to note that not all confirmatory
reagents lend themselves to performance of a confirmatory mixing test; however, confirmatory assays in
paired systems (dRVVT, SCT, dPT) can be used, if appropriately validated. Use of a confirmatory mixing
test (1:1 dilution) may increase specificity where there is a coexisting cause of clotting time
prolongation.29,179,259,269 This is because a non-LA abnormality can compromise testing on undiluted (neat)
plasma. The mixing test should be performed with the same assay that showed the prolongation (ie,
dRVVT confirmatory mixing test is performed because dRVVT confirmatory assay is prolonged). If
clotting times of a 1:1 confirmatory mixing test “correct,” there is a high degree of suspicion for a factor
deficiency (congenital or acquired, such as VKA) or that LA is present concomitantly with VKA. A
prolonged 1:1 confirmatory mixing test suggests the presence of an inhibitor other than LA or a very
strong LA that overwhelms the bypassing effect of the added PL.
12.1.4 Mixing Test and Integrated Assays
The HPNT and APTT lupus ratio204 tests are considered integrated assays because they directly
incorporate a mixing test within both screening and confirmatory procedures. This is in contrast to paired
tests where the mixing process is performed separately (“off-line”). With integrated assays, both
screening and confirmatory steps dilute (mix) patient PPP with NPP and both steps are performed at the
same time. The incorporation of NPP makes a separate mixing test unnecessary.28,197
12.2 Normal Pooled Plasma
The quality of an NPP is considered by many to be paramount to a successful mixing test. Considerations
include adequate coagulation factor activities, sufficiently platelet poor, “home-made” vs a commercial
preparation, and frozen vs lyophilized storage. An undisputable requirement is that the clotting times of
an NPP fall well within the RI of the reagent test system that is used (eg, APTT, dRVVT).
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Lyophilized normal plasmas prepared specifically for use as NPP can be used270 but care must be taken to
completely dissolve these products. This is particularly true when using certain analyzers that are very
sensitive to residual precipitated protein. Lyophilized normal plasmas that are manufactured specifically
as test controls must not be used because various buffers and stabilizers are added to these products.
Nonlyophilized (frozen) NPP may be a more suitable alternative because it most closely resembles the
plasma with which it will be mixed, limits potential for false-positive results, and avoids potential
confusion in the laboratory when considering choices in lyophilized materials.270 NOTE: Previously run
patient samples must not be used because normal PT and APTT values do not imply normalcy (ie, results
may be “normal” due to elevation of acute phase proteins).
This guideline recommends the following in evaluating an NPP to be used for a mixing test:
 “Home-made” NPP should be prepared from a minimum of 20 normal individuals (equal ratio
male/female) in order to statistically yield > 80% of all coagulation factors. (See CLSI document
H47135 for additional information.)
 Platelet count should be < 10 × 109/L. This can be determined by using an automated cell counter or
found in documentation provided by a commercial vendor for its product.
 Activities for coagulation factors (FII, FV, FVII, FVIII, FIX, FX, FXI, and FXII) should be ≥ 80%.271
This can be determined by performing individual factor assays on an NPP or found in documentation
provided by a commercial vendor for its product.
 The absence of LA in NPP should be documented by performing LA tests that are routinely used by
one’s laboratory.
 NPP that is frozen should be stored at −70°C or below. Lyophilized NPP should be stored according
to the manufacturer’s instructions.
12.3 Calculation of Test Results
Equally critical to the performance of a mixing test is determining the calculations used for subsequent
test interpretation. Historically, the most common method has not required a calculation. Simply
“correction” or lack thereof was determined by use of an in-house-established RI for the assay to which
the mixing test is related (eg, a dRVVT screen RI serves as a point of reference for a dRVVT mixing
test). However, numerous variations for the target correction point have been considered, such as
correction to within upper limits of the RI defined as + 2 SDs, + 3 SDs, or within five seconds of the + 2
SD limit.137 Moore et al. have shown that an RI established for the specific mixing test assay (ie, dRVVT
screen mixing test) is more sensitive than using the parent test (dRVVT screen) RI.260 This guideline
recommends that RI be established specifically for each assay mixing test (eg, dRVVT screen mixing test,
SCT screen mixing test, dPT screen mixing test) (see Appendix B).
This guideline supports two methods for calculating and reporting a mixing test result:
 A 1:1 mix test normalized ratio

 The ICA
Either is acceptable irrespective of which screening/confirmatory assay is used and must be validated for
each specific assay (eg, APTT screen mixing test, dRVVT screen mixing test, dRVVT confirmatory
mixing test).
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12.3.1 Mixing Test Normalized Ratio
Results are expressed as clotting times in seconds (s). This guideline recommends that results for any
mixing test be calculated and reported as a normalized ratio (see Appendix F) with normalization to the
mean of the RI for the specific mixing test. A ratio is calculated:
Screen 1:1 Mix Test Normalized Ratio = Screen 1:1 Mix (s) / Screen 1:1 Mix Mean of RI (s)
or
Confirm 1:1 Mix Test Normalized Ratio = Confirm 1:1 Mix (s) / Confirm 1:1 Mix Mean of RI (s)
Historically, a mixing test result has been normalized to a neat NPP performed in like manner on the same
day (eg, an APTT 1:1 mixing test result is divided by a neat NPP APTT result). The rationale for using
the NPP on the day of testing is to take into account day-to-day variability. However, this has already
been done if an RI was established appropriately (eg, five samples per day for eight days as noted in
Appendix B). Hence, using the mean of an RI instead of an NPP in the denominator reflects this type of
variation. Moreover, the target correction point is potentially not gauged against a particular NPP value
(correction to within five seconds of an NPP value, 10% of an NPP value, or lack of correction at > 15%
of NPP value)137 but instead against the RI established for the specific mixing test in question.
12.3.2 Mixing Test Index of Circulating Anticoagulant
Unlike comparisons to a “normal” parameter (as noted above), the ICA takes into account the degree to
which a mixing test corrects relative to the patient’s initial prolonged clotting time. The ICA, also known
as the Rosner Index,62 is based on a 1:1 mixture and can be applied to any screening assay provided it is
validated for each specific test. In other words, a cutoff value determined for an APTT screening test
cannot be used for a dRVVT screening test. The formula (see Appendix F) is:
[Screen 1:1 Mix (s) − Screen 1:1 RI Mean (s) / Screen Patient (s)] • 100
Other calculations have been used for a mixing test, such as a percent correction of clotting times,272,273
but are not supported by this guideline.
12.4 Limitations of Mixing Test
The mixing test is affected by the same preexamination and examination issues that affect its parent test
system. Reagent responsiveness to LA (too responsive or not sufficiently responsive), particularly with
APTT reagents and reagents used in LA screening assays, is critical to the effectiveness of a mixing test.
As noted above, the quality of NPP is critically important because a factor deficiency cannot be corrected
if the product used for making the correction is not in and of itself normal. When performing a mixing test
with any assay (preliminary APTT or PT, LA screening or confirmatory assays), a good QC check, before
preparing test mixtures, is to test the NPP with the specific assay to be used in order to ensure the
integrity of the NPP.
The inherent design of a mixing test creates a significant limitation—that of a dilutional effect. A 1:1
mixture dilutes the concentration of the LA antibody, which could potentially lead to false-negative
results if the antibody is weak. Moreover, this can be problematic in patients taking VKA with or without
the presence of an LA. Kaczor and colleagues suggested that with a classical APTT, a 4:1 mix (four parts
patient PPP to one part NPP [see Appendix D, Figure D12b]) was more sensitive because a weak
inhibitor could potentially be masked (diluted) due to the amount of NPP present in a 1:1 mix.261 An
important caveat to the 4:1 mix is that the level of coagulation factors provided by the 20% NPP may not
be sufficient to correct a mild factor deficiency (similar to factor levels seen with VKA) and thereby yield
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a false-positive mixing test result. Thom and colleagues observed a high percentage of false-negative
results (APTT and dRVVT) in known LA patients, who were or were not receiving VKA, and concluded
that simply because NPP corrected an abnormal screening test, this alone should not exclude further LA
testing.257 Other workers, using an assortment of assays, similarly demonstrated that the inability by a
weak LA to show inhibition is dependent on the dilution effect of a 1:1 mixing test.260,274 Tripodi and
colleagues275 compared the SCT and dRVVT assays, both with no associated mixing test, to the HPNT,
which integrates a mixing test in both the screening and confirmatory steps of the test. Both the SCT and
dRVVT assays showed sensitivities and specificities comparable to the HPNT, implying that a mixing
test may not be necessary. Though collectively these works assess the limitation of this assay and thereby
question the effectiveness of a 1:1 mixing test, others have continued to support its inclusion in an LA
panel.169,259
High dose VKA can lead to a noncorrecting mixing test.10 DTI can cause false-positive mixing tests.
Limited data are available for direct FXa inhibitors and therefore results of mixing tests should be
interpreted with caution.
Certain coagulopathies that affect clot-based testing may also interfere with LA testing. Any single,
severe factor deficiency or deficiencies of multiple factors (acquired or congenital) may not achieve full
correction of a 1:1 mixing test back into the RI. Specific factor inhibitors, such as those to FV, FVIII, or
FIX, may also mimic an LA.137,154,190,207 Young children (0 to 12 months old) have lower factor levels,
which may cause an abnormal LA screening test that may not correct with a mixing test.276,277
13 Postexamination Issues
This guideline recommends that data derived from LA assays be accompanied by an interpretation. What
type of interpretation is to be done, by whom, and how this is to be accomplished significantly affects the
quality of the interpretation. This guideline does not consider “canned” computer comments as
interpretation but rather as test disclaimers. Previous sections of this document have presented tools to
laboratorians, laboratory directors, and clinicians to help with the interpretation of LA test data.
Interpretation of LA test data is performed at various levels within the laboratory. The laboratorian is
relied upon to interpret data from each test as it is performed. This individual should check test results to
ensure they are not aberrant due to preexamination or examination concerns, that all QC and QA
parameters (see Section 14) have been satisfied, and to question how test results relate to each other (ie,
does each test result make sense relative to the other?). This first line of interpretation will permit
immediate retesting in order to reduce interpretation errors. Secondly, the overall laboratory interpretation
of all LA testing should be performed by a qualified individual (eg, laboratory director or delegate) who
has knowledge of specific assays comprising the laboratory’s LA panel, understands the limitations of
these tests as noted throughout the limitations sections of this document, and considers all testing data in
total to reach a laboratory diagnosis. Thirdly, clinicians will make a clinical interpretation when linking
the laboratory diagnosis with their patients’ overall clinical status. It is the clinician and not the laboratory
who will determine a diagnosis of APS.
Section 6.3 addresses how a laboratory establishes RI/cutoff values. These limits permit assignment of
data as “LA not detected” (a result is below the RI/cutoff value for a specific test) or “LA present” (result
is equal to or above RI/cutoff value for a specific test). Though LA activity is not observed in a particular
test, this does not completely exclude the presence of LA because the test itself may not be sufficiently
robust to capture this activity. Therefore “LA not detected” is the preferred term and is not synonymous
with “negative.” Where LA activity is detected, the preferred term is “LA present” and not “positive.”
This guideline acknowledges that each test result when compared to its specific RI/cutoff will be
classified as either “LA not detected” or “LA present”; however, consideration of all tests in total may not
permit such a clear distinction. Therefore, this guideline supports the use of the term “indeterminate.”
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Section 7 draws awareness to the numerous preexamination variables that affect clot-based assays in
general and LA assays specifically. It is important to note that patients themselves are such a variable.
The use of preliminary tests (see Section 8) such as the APTT, PT, and TT challenge the integrity of the
sample and begin to draw attention to potential interferences that may affect LA testing and results. If
interferences (see Appendix E) such as VKA, DTI, FXa inhibitors, and UFH are discovered, then this
guideline strongly suggests that testing for LA not be performed because test interpretation can be
difficult and risks identifying individuals as having LA present when they do not. Section 9 discusses
principles for LA tests and categorizes these assays according to the coagulation pathways (see Appendix
C) that they address. This information is important in light of Criterion/Recommendation B, which states
that two tests, representing different principles and coagulation pathways, must be used to screen for LA.
Sections 10 to 12 present various assays used to screen and confirm for the PL nature of LA as well as
demonstrate its inhibitory activity. Calculations, recommended for normalizing test results, as well as
limitations of these tests are also available in these sections. Each elevated screening test must be
accompanied by its confirmatory test, and, where necessary, a mixing test. Confirmatory test results do
not always return to the RI when LA is present, so LA activity may be clearer in one test system
compared to another,10,28 and multiple test positivity may represent a greater thrombotic risk.251 Therefore,
all elevated screening tests must be fully evaluated. Criterion/Recommendation E notes the impact that
interfering factors can have on testing and subsequent interpretation (see Appendix E). Agents such as
VKA, heparin, DTI, and FXa inhibitors can give rise to false-positive interpretations of screening and
confirmatory assays,87,100,189 further emphasizing the need to employ preliminary assay results as an
integral component of LA testing interpretation. Factor assays should be ordered and performed (using
three or more dilutions of patient plasma and an APTT reagent that is unresponsive to LA) whenever a
specific factor deficiency or factor inhibitor cannot be excluded.
13.1 Interpretation of Preliminary Examination Assays
Preliminary examination assays (PT, APTT, and TT) reveal crucial information about whether the
patient’s coagulation profile is normal, may contain an LA or other inhibitor, or may suggest therapeutic
anticoagulation or factor deficiencies. In order to facilitate informed interpretations, LA assays must be
interpreted in conjunction with these preliminary examination tests because they provide invaluable
information about which LA assay systems may or may not be affected by coexistent abnormalities, or
signal that LA testing may be unreliable, or that other tests, such as factor assays, may be
informative.27,28,89 A normal coagulation screen does not exclude the presence of LA, even when
employing an APTT reagent shown to be responsive to LA.27,89,107,152 Therefore, if clinical suspicion is
high for APS and an initial APTT is within RI limits, the laboratory should proceed with LA testing. On
the other hand, the potential presence of abnormalities other than LA does not necessarily preclude
proceeding with LA testing (eg, heparin levels within the neutralizing capacity of reagents that contain
heparin-neutralizing agents). In cases where the PT and TT are normal, LA assays can be interpreted
knowing that little if any interfering substances were present. However, some interfering substances may
manifest in LA assays but not coagulation screening tests.
The following tables in Sections 13.1 to 13.4 walk the reader, step-by-step, through the analysis of one
sample using individual tests under the four main testing categories: 1) preliminary, 2) screening, 3)
confirmatory, and 4) mixing. Each table shows the individual test conclusion that a laboratorian would
reach upon performing the test. The commentaries that follow each table draw attention to considerations
that affect testing and provide an interpretation for each of the four testing categories. These four
interpretations are considered together to render an overall interpretation of an LA panel (see Section
13.5).
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Results (Seconds or Individual Test

LA Panel Ratio) RI/Cutoff Conclusions
Preliminary Examination Assays
 PT (s) or PT-INR 11 10–12 N
 APTT (s) 50 30–40 *
 TT (s) 10 9–11 N
*
“Up” arrow () indicates “increased.”
Abbreviations: APTT, activated partial thromboplastin time; LA, lupus anticoagulant(s); N, normal; PT, prothrombin time; PT-
INR, prothrombin time-international normalized ratio; RI, reference interval(s); s, seconds; TT, thrombin time.
Assessment of results from the above example table:
 PT is within RI indicating no interferences from pharmacological agents (VKA or DTI) or

extrinsic/common pathway factor deficiencies (FVII, FX, FV, FII, and fibrinogen). FXa inhibitors
cannot be completely excluded because some PT reagents are relatively insensitive to rivaroxaban,
and fondaparinux often does not prolong the PT.
 APTT (routine reagent) prolongation can be due to: pharmacological interference (eg, UFH), intrinsic
pathway factor deficiencies (contact factors [FXII, PK, HMWK] or FXI, FIX, FVIII), or LA. Check
TT.
 TT is within RI, indicating that APTT prolongation is most likely not due to UFH or DTI.
 Interpretation of preliminary assays: Sample integrity permits further testing; APTT prolongation is
due to LA, intrinsic pathway factor deficiencies, or inhibitors specific to those factors.
13.2 Interpretation of Lupus Anticoagulant Screening Assays (Criterion/Recommendation B)
Testing in an LA panel is performed in sequence, starting with two screening tests, each challenging a
different coagulation pathway. This guideline supports the use of the APTT (using an APTT reagent that
is responsive to LA) and dRVVT as first-line screening tests. If both screening test results are within
locally derived RI/cutoff values, no further testing is required and the report can be issued with a
comment indicating that LA was “not detected.” However, if the clinical suspicion of APS remains (see
Section 7.1.2), screening tests should be repeated on a fresh sample and performance of the tests with
alternative reagents, or second-line assays, could also be considered.210 Elevation of at least one PL-
dependent screening test above the cutoff of a locally derived RI is the first step in detection of LA in a
test plasma.27,28,86,89 However, at this stage of the diagnostic pathway, the presence of LA is but one of
many possible causes of clotting time prolongation. LA are detected by inference based on data indicating
their behavior in the medley of screening, confirmatory, and mixing test data, so an elevated screening
test is potentially suggestive of the presence of an LA, but requires both the confirmatory test(s) and
possibly mixing test(s) to demonstrate PL dependence and inhibitory action, respectively. If one or both
screening assays are prolonged, then confirmatory tests must be performed using the same assay principle
as the screening test(s) that was initially found to be abnormal.
Antibody and reagent heterogeneity mean that not all LA will necessarily react equally between test
systems, and some may react in one screening test but not another. A prolonged clotting time in an LA
screening test where the preliminary assays are normal is suggestive of an LA, although minimal elevation
could represent a weak LA, an antibody that reacts poorly in that test system, or merely a statistical
outlier. Knowledge of test principles is invaluable when non-LA abnormalities are present. For example, a
patient with an FVIII or FIX inhibitor could compromise APTT-based LA testing, but other assays, such
as dRVVT and TSVT, would be unaffected and their results could be interpreted at face value.
Alternatively, a patient in whom therapeutic levels of UFH have been achieved can often be tested if the
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reagents in a specific test system contain a heparin neutralizer. It is important to recognize that a patient
may have an LA that is not reactive in the test system used and masked in a test system that is
compromised.
Elevated levels of FVIII can mask the presence of LA in an APTT-based system. If values for preliminary
APTT and/or APTT used for LA screening (responsive to LA) are both at or below the lower limits of
their RI, elevated FVIII, sample activation, or a statistical outlier can be suspected.177 An APTT screening
mixing test may dilute the FVIII and allow an LA to manifest, although weak LA may themselves be
diluted and thus missed. If the dRVVT screening test is normal, a third LA assay could be considered.
Concern has been expressed that the risk of false-positive test results is increased to an unacceptable level
if more than two screening tests are performed.28 However, while use of more than two screening tests
may well result in more positive individual screening test results, application of the confirmatory test(s)
will not lead to more positive overall interpretations. Instead, genuine LA that were unreactive in first-
line assays may be identified. 175,176,178,181,210 Some laboratories choose to routinely perform three assays,
each covering the intrinsic, extrinsic, and common pathways to minimize this problem.210,238
Results Individual
(Seconds or Test
LA Panel Ratio) RI/Cutoff Conclusions
Screening Assays
 APTT Screen – Intrinsic Pathway [Independent] Test #1
APTT Screen (s) 53 31–39 *
APTT Screen Normalized Ratio (RI Mean = 35) 1.51 < 1.20 ABN
 dRVVT Screen – Common Pathway [Paired] Test #2
dRVVT Screen (s) 56 33–43 
dRVVT Screen Normalized Ratio (RI Mean = 38) 1.47 < 1.18 ABN
*
Abbreviations: ABN, abnormal; APTT, activated partial thromboplastin time; dRVVT, dilute Russell’s viper venom time; LA,
lupus anticoagulant(s); RI, reference interval(s); s, seconds.
 Intrinsic pathway screening test result is prolonged using an APTT reagent responsive to LA yielding
a normalized ratio that is greater than the cutoff. Check independent confirmatory test (PNP).
 Common pathway screening test result is prolonged yielding a normalized ratio that is greater than
the cutoff. Check paired confirmatory test (dRVVT confirm).
 Interpretation of screening assays: Both screening assays are abnormal suggesting the possible
presence of LA. Check confirmatory tests for both assays.
13.3 Interpretation of Lupus Anticoagulant Confirmatory Assays

(Criterion/Recommendation C)
Elevated LA screening tests should be followed by confirmatory assays to determine or exclude PL

dependence. The addition of altered concentrations and/or composition of PL attenuate the inhibitor
effect, thus shortening a clotting time (Criterion/Recommendation C). The screening test(s) that was
elevated is repeated with a confirmatory assay(s) using the same assay principle. For paired test systems
(dRVVT, SCT, dPT) the companion confirmatory reagent is used whereas independent tests (APTT,
KCT) will use an independent confirmatory test. If a normalized ratio for a screening assay is below the
cutoff value, then it is not obligatory to perform a companion confirmatory test. However, some paired
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tests (eg, SCT) and the integrated test, HPNT, perform screening and confirmatory assays on every
sample and concordance/nonconcordance in the context of PL dependence is assessed mathematically.
This is done whether or not the screening test value is abnormal. Advantages of this approach are that
confirmatory test results are immediately available and “weak” LA that prolong clotting times above an
individual patient’s baseline, but not above the RI, are more likely to be detected, in part, because
nonconcordance between both tests may reveal the LA where the screening test alone would
not.28,101,102,211
The non-PL-dependent abnormalities (see respective test sections and Appendix E) that elevate LA
screening test results will, in most cases, generate similarly elevated results in confirmatory assay clotting
times or normalized ratios. These similar elevations could indicate an isolated factor deficiency or non-
PL-dependent inhibitor that could also mask an LA. Although a screening test in isolation may have low
specificity, interpreting the results in conjunction with a confirmatory assay facilitates more accurate
diagnosis.
Some genuine LA can resist the swamping effects of confirmatory reagents such that a significant
correction is not realized. Therefore, when LA screening tests are elevated, there is no evidence of other
coagulation abnormalities, and there is insignificant correction of the confirmatory assay, and the
presence of such LA can be suspected. These antibodies can be characterized as LA by increasing the
concentration of PL in the confirmatory assay236,278,279 and/or employing other second-line assays
(Textarin time or TSVT as LA screening assays accompanied by Ecarin time as the confirmatory
assay).211,213,215,236
Clotting times (Tube #2) derived from PNP (independent confirmatory assay) and HPNT (integrated
assay) show “correction” by shortening but yielding a delta (“correction”) that is greater (larger number)
than a specified cutoff value. Clotting times derived from confirmatory assays in paired test systems show
“correction” by shortening but yielding normalized confirmatory ratios that are less than a specified cutoff
value (see example table below).
The manner in which screening and confirmatory data are calculated, particularly for dRVVT, has a
significant impact on LA interpretation and may affect sensitivity and specificity more than the choice of
kit.86 When assessing screening to confirmatory ratios for paired test systems, application of recommended
calculations (see Appendix F) such as the normalized ratio or percent correction of normalized ratio
permit distinction between significant and insignificant correction of screening results by confirmatory
results. If numbers derived from these calculations are less than the locally established cutoff limits, then
LA is “not detected” and no further LA testing is necessary. If calculated values are equal to or greater
than cutoff limits, then interpretation should proceed to screening and/or confirmatory mixing tests in
order to help reveal inhibitor activity. In a paired test system, a confirmatory mixing test is performed if
the confirmatory assay is prolonged. This may possibly aid in differentiating inhibitor activity from factor
deficiencies.
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Results Individual
(Seconds Test
LA Panel or Ratio) RI/Cutoff Conclusions
Confirmatory Assays
 PNP Confirm (Independent Test for APTT Screen)
Tube #1 Plasma/Saline (s) 48 29–37 *
Tube #2 Plasma/PL (s) 45 30–38 ABN
PNP delta (s) 3 <4 N
 dRVVT Confirm (Paired Test for dRVVT Screen)
dRVVT Confirm (s) 33 32–41 N
dRVVT Confirm Normalized Ratio (RI Mean = 0.90 < 1.15 N
36.5)
 dRVVT Paired Test Calculations (use one or other)
dRVVT Screen/Confirm Normalized Ratio 1.63 < 1.15 ABN
dRVVT Screen/Confirm Normalized Ratio % 38.8 < 10% ABN
Correction
*
Abbreviations: ABN, abnormal; APTT, activated partial thromboplastin time; dRVVT, dilute Russell’s viper venom time; LA,
lupus anticoagulant(s); N, normal; PL, phospholipid(s); PNP, platelet neutralization procedure; RI, reference interval(s); s,
seconds.
 For PNP: Tube #1 is prolonged (48 s) and also prolonged (but slightly less so) compared to baseline
screening APTT (53 s) using the same APTT reagent. Tube #2 with added PL did not cause clotting
time to shorten. Delta seconds (3) indicates that shortening of clotting time in Tube #2 relative to
Tube #1 is due to dilution effect and not PL dependence.
 For dRVVT confirm: Excess PL in confirm reagent caused clotting time to shorten to within RI (from
56 to 33 s). Value is less than cutoff and PL dependence is confirmed.
 Both normalized ratio and percent correction of normalized ratio are greater than cutoff indicating PL
dependence.
 Interpretation of confirmatory assays: The APTT confirmatory assay (PNP) failed to show that the
abnormality is PL dependent; therefore, it is necessary to perform an APTT screen mixing test. The
dRVVT normalized screen to confirm ratio showed PL dependence and the confirm ratio was within
the RI; therefore, no further testing is required with the dRVVT test system.
13.4 Interpretation of Lupus Anticoagulant Mixing Tests (Criterion/Recommendation D)
By definition, LA antibodies are inhibitory in nature so diagnostic specificity is improved if this

characteristic can be established. If an inhibitor is present, the clotting time in the mixing test should be
elevated above the clotting time the mixture would have had in the absence of the inhibitor. When
interpreting a mixing test, it is important to be cognizant of the unavoidable twofold dilution of any LA
antibody that may be present. This can lead to a false-negative interpretation of the test but does not
exclude the presence of an LA if screening and confirmatory results on undiluted plasma are clear cut and
there are no interfering factors.10,28,90,138,154,257,260,262,280-282 Hong and colleagues have shown that the
presence of LA when using only a two-step procedure (screening and confirmatory tests) showed higher
thrombotic risk than using the traditional three-step procedure (screening, mixing, and confirmatory
tests).282 In view of the dilution effect in mixing tests, as well as other limitations noted in Section 12.4, a
positive result can be diagnostic, but negative mixing tests in a patient with another abnormality cannot
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exclude the presence of LA. (See examples 7 and 8 in Appendixes G1 and G2.) Therefore, this guideline
has placed more emphasis on demonstrating the PL dependence of the antibody instead of its inhibitory
action, thereby placing the mixing test as third in the testing sequence. However, this guideline continues
to support the use of the mixing test if discrepancies occur between screening and confirmatory tests, to
increase diagnostic specificity, and to enhance detection/interpretation when there are coexisting
abnormalities. In contrast, this guideline does not encourage performance of a screening mixing test if
screening and confirmatory testing indicate the presence of LA.
A mixing test for an LA screening assay is performed if both the screening test result is abnormal and the
confirmatory test does not show PL dependence. A normal screening mixing test suggests that a factor
deficiency (congenital or acquired) may have caused the abnormal LA assays. On the other hand, as noted
above and in Section 12.4, a weak inhibitor may have been masked. For paired test systems, mixing tests
employing screening and confirmatory assays may: 1) correct a factor deficiency and reveal a masked
LA, 2) both return to their RI and suggest an isolated factor deficiency (with recognition of the dilution
effect on a weaker LA), or 3) both remain similarly elevated and suggest a non-PL-dependent inhibitor or
the presence of a “strong” LA.
Results Individual
(Seconds RI/ Test
LA Panel or Ratio) Cutoff Conclusions
Mixing Tests
 APTT Screen 1:1 Mixing Test
APTT Screen 1:1 Mixing Test (s) 46 31–37 *
APTT Screen 1:1 Mixing Test Normalized Ratio (RI Mean 1.35 < 1.15 ABN
= 36.5)
*
Abbreviations: ABN, abnormal; APTT, activated partial thromboplastin time; LA, lupus anticoagulant(s); RI, reference
interval(s); s, seconds.
 APTT screen mixing test (using LA-responsive reagent) failed to “correct” indicating the presence of
LA (either in conjunction with a factor deficiency or LA is “strong”). Factor assays should be
performed to distinguish LA from other causes of prolonged clotting times, such as factor deficiencies
or specific factor inhibitors that may mask, mimic, or coexist with LA.
 Interpretation of mixing tests: APTT screen mixing test showed inhibitory effect of LA on NPP.
Intrinsic pathway factor deficiencies should be excluded (PT is normal, excluding extrinsic pathway
factor deficiencies and dRVVT confirmatory assay returned to RI, suggesting that common pathway
factor levels are within normal limits).
13.5 Interpretation of Lupus Anticoagulant Panel (Criterion/Recommendation F)
Following the step-by-step review of all raw data and calculations, a qualified individual (eg, laboratory
director or delegate) who has knowledge of specific assays comprising the laboratory’s LA panel will
need to bring together this information and provide an overall interpretation and laboratory diagnosis.
For the example provided in Sections 13.1 through 13.4, an overall interpretation could read as follows:
“The laboratory diagnosis is consistent with the presence of a lupus anticoagulant by dRVVT and
APTT analysis, as follows: Preliminary assays showed a prolonged APTT and that the sample was
appropriate for further lupus anticoagulant testing. Phospholipid dependence was accentuated with
low phospholipid reagents when using both screening assays (APTT and dRVVT) and attenuated with
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high phospholipid confirmatory dRVVT reagent. PNP confirmatory assay failed to indicate
phospholipid dependence. APTT screening mixing test showed the inhibitory effect that a lupus
anticoagulant had on normal pooled plasma. Lupus anticoagulant test panel should be repeated at or
beyond 12 weeks to determine persistence of lupus anticoagulant as part of the evaluation for
antiphospholipid syndrome.”
As noted in the above example, the interpretive comment should alert the patient’s physician when to
repeat LA testing. For patients in whom LA is not detected but there is a high clinical suspicion for APS, it
may be appropriate to retest on a fresh sample and/or perform alternative LA and aPL assays. If LA is
present in a patient sample (as noted above), then repeat testing is suggested at or beyond 12 weeks to
determine if LA is persistent. If LA testing results, in total, are inconclusive, then an “LA indeterminate”
interpretation suggests that LA cannot be confirmed or excluded without repeat testing on a new sample.
Appendix G provides 11 examples of succinct interpretive comments (see Appendix G2) and the rationale
for those comments based on patient data (see Appendix G1). The tables permit one to compare and
contrast data and interpretation for different types of samples, including those with “LA not detected,”
“LA present,” “LA indeterminate,” and others in which interfering substances were found. All testing is
shown for completeness in the data table (see Appendix G1).
13.6 How to Report Test Results (Criterion/Recommendation F)
Reports should contain all relevant data from screening, confirmatory, and mixing tests. The raw data
should be accompanied by RI/cutoffs and an informative interpretive report. The final interpretation
should clearly indicate one of the following: LA present, LA not detected, or LA indeterminate. It should
be clear from the wording that the interpretive comment is an assessment of the laboratory data for the
presence or absence of LA and not a diagnosis of APS or any other disease process. Follow-up and/or
additional tests can be suggested when considered to be useful or necessary to enhance diagnostic
pathways. Solid-phase assays for PL-dependent antibodies (eg, aCL and/or aβ2GPI) must be
recommended as part of an evaluation for APS. Moreover, if LA is present, a recommendation must be
made to repeat the test panel at or beyond 12 weeks to determine persistence of LA as part of the
evaluation for APS.
14 Quality Control and Quality Assurance
14.1 Reagent Lot-to-Lot Number Verification
The method validation process (see Section 6) defines the characteristics of a test for the reagent lot
numbers used at the time of the validation. Most importantly, RI and cutoff values are defined during this
process for specific lot numbers of reagents. Eventually, these initial lot numbers of reagents will be
depleted and new lot numbers will be needed. Each lot number of new reagent should be checked to show
that the RI/cutoff values are still appropriate for each test. Two checks are used: 1) verification of
RI/cutoff value; and 2) method comparison between lot numbers.
Verification of an RI entails running 20 samples considered to represent the reference population. The
current RI is considered verified as long as two results or fewer from these 20 samples fall outside of the
current RI (see CLSI document EP28).83 If more than two results fall outside the current RI, then 20 more
samples should be run and a new RI established (see Section 6.3). If a minimum of 40 samples do not
yield an appropriate RI, then 120 normal samples will need to be used as noted in CLSI document EP28.83
Subsequent to verification and/or establishment of a new RI, a method comparison is performed wherein
40 samples (normal and representative abnormal samples, as discussed in Section 6.5) should be tested
using both the old and new lot numbers of reagents (see CLSI document EP0973). Special consideration
should be given to samples at the critical cutoff values. Patient samples can be collected during the year
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and kept in a −70°C freezer to be used for lot conversion comparison studies. The comparison studies
should preferably be run simultaneously (but if this is not possible, as soon as possible) to exclude a
sample stability variable into the calculations. The comparison data should be plotted into a scatter and
bias plot as described in CLSI document EP09.73 Data from a method comparison study should yield
identical diagnostic outcomes, ie, if LA is found not detected or present in a sample when using an old lot
number of reagent, then a new lot number should give a similar finding.
14.2 Internal Quality Control
As for all assays within hemostasis, the use of internal QC is critical to ensure the appropriateness of test
results. For LA tests, suitable controls will include normal and abnormal LA material, with the latter
comprising samples both near and distant to a respective cutoff value for a particular test. Although
commercial assay systems may include “in-kit” QC materials, there is added value in the use of additional
external (non–kit supplied) QC material with all assays.283 The LA positive control set should include
material that generates results around the clinically important cutoff value for each assay. The selection of
QC material with these properties is important for detecting problems with an assay at these clinically
important decision points, which may determine the absence or presence of LA by a particular assay. It
may be necessary to employ more than one commercial LA-positive QC preparation because not all
products will necessarily generate positive results in every reagent/analyzer pairing. QC material can be
obtained as pools of in-house positive specimens or as commercial material diluted to provide optimal test
values.
The use of non–kit supplied QCs will also enable the monitoring of interbatch (interlot) variation,
generation of information regarding comparability with previous lots, as well as information regarding
comparability of results when changing between different commercial kits. The medium- to long-term
picture of ongoing assay performance provided by these external controls in their assays cannot typically
be obtained by using only kit-supplied controls, because these are generally optimized (and changed) for
each new batch/lot of commercial kits.
14.3 External Quality Assessment
All laboratories are expected to participate in EQA programs for their tests/assays, and such programs are
currently available for most geographic regions. Participation in such activity is usually mandatory for
ongoing laboratory accreditation.283-285 These EQA programs also provide another means to monitor local
test activity, a comparison base with peer laboratories, and educational and technical support. Although
the results from these EQA programs and similar evaluations have highlighted ongoing problems in LA
test performance,283-286 they also provide some evidence of improvement related to LA testing by
laboratories that have ongoing EQA participation accompanied by correction and amendment of any
suboptimal practices.
In general, laboratories should aim to follow the median trends of peer laboratory–reported values for
EQA test material as used in their assay or comparable assays. Outlier status usually identifies problems
with laboratory testing, although outliers can occasionally represent more clinically relevant results. The
latter can occur when the EQA data are skewed by test results from a dominant but inferior assay process,
or when advances in testing are starting to emerge and test data are skewed by older and inferior (but still
dominant) methodologies.285 However, outlier status more often indicates test problems requiring
rectification. Guidance on laboratory EQA performance can be obtained from the EQA provider.
Laboratories should pay particular attention to EQA data related to results of “weak” LA samples (values
are near an assay cutoff value). For example, review of methodology or cutoff values is particularly
indicated where laboratories consistently report not detected LA results for EQA-tested weak LA
samples, because this has significant implications for reporting of clinical samples. Accordingly, it is
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recommended that EQA providers include the distribution of a weakly positive LA sample to participants
on a regular basis (at least once a year, but preferably twice).
15 Harmonization of CLSI Lupus Anticoagulant Guideline With Other

Guidelines
Guidelines for LA testing are available from various national organizations9,29,30 and internationally from
the ISTH.25-28 LA testing has improved over the nearly 30 years since the first guideline was put forth and
changes in therapeutics alone have rendered older guidelines less effective. Therefore, this guideline
harmonizes more closely with the recent ISTH SSC 2009 guidelines than previous ISTH publications.
Table 3 shows where the two guidelines differ and the reasoning for those disparities.
Adherence or nonadherence to guidelines224,238,287 may depend, in part, on availability of assays to a

laboratory, financial constraints, and physician ordering practices. Additionally, the popularity
(acceptance) of particular assays may drive their inclusion into guidelines and/or a laboratory’s LA panel,
though lesser known assays may be comparable or better. Clearance (approval) by governmental agencies
for certain assays may drive the marketplace and create a potential for noncompliance to guidelines. It is
hoped that this guideline has presented information in a succinct, practical, and easy-to-understand
format, thereby helping to standardize an approach to LA testing, gain acceptance in practice, and
improve testing quality.
Table 3. Contrasts Between ISTH SSC 2009 and CLSI H60 Guidelines
ISTH SSC 2009 CLSI H60 Rationale*
Defines integrated test system as one in
which no step can be performed
Defines integrated tests as assay independently (screening, confirmatory, and
systems in which screening and mixing test are performed simultaneously in Sections
confirmatory are performed in parallel one test system, such as the HPNT); paired 4, 9, 10, 11,
and mixing test(s) may or may not be assays consist of tests of like principle that 12
performed. are paired as screening and confirmatory
assays (ie, dRVVT) and performance of a
mixing test is optional.
Determines RI/cutoff values at 99% Determines RI/cutoff values at + 2 SDs Section 6.3;
quantile (nonparametric). (parametric) for each test in an LA panel. Appendix B
Limits APTT activator to silica because Does not restrict type of APTT activator that Sections
“ellagic acid is insensitive for LA.” can be used for APTT reagent. 8.1, 9.1.1
Recommends that both APTT and dRVVT
Recommends that dRVVT should be
(in no particular order) be performed as first-
the first test considered and only Sections
line screening assays, but does not exclude
dRVVT and APTT (LA “sensitive”) 10.1, 13.2
use of other assays as long as two different
are to be used as screening assays.
coagulation pathways are considered.
Does not recommend use of the Does not restrict or exclude use of second-
following tests: dPT (variability in line assays such as dPT (commercially
thromboplastin reagents), snake venom available), snake venom assays Sections
assays (no standardized commercial (commercially available), KCT (low 9, 10
assays available), KCT (poorer turbidity kaolin is commercially available
reproducibility due to kaolin turbidity). for automated analyzers).
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Volume 34 H60-A
Table 3. (Continued)
ISTH SSC 2009 CLSI H60 Rationale*
Order of testing algorithm: screening,
Order of testing algorithm: screening, confirmatory, mixing
mixing, confirmatory
Assigns a lower priority to a mixing test Sections
Suggests the presence of LA if an because of its limitations; prioritizes the 10, 11, 12;
elevated result is obtained in the demonstration of PL dependence of the Appendix A
mixing test. antibody (screening and confirmatory assays)
over showing inhibitory action of LA (mixing
test).
Clarifies that confirmatory assays be
performed using the same assay type that
Implies but does not clearly indicate
resulted in a positive screening test and if
when confirmatory assays should be Section 11
both screening assays were prolonged, then
performed.
confirmatory assays should be performed on
both tests.
Sections
Results should be expressed as ratios Results should be expressed as ratios
6.3.1, 10, 11,
(normalized) of patient to NPP for all (normalized) of patient to the mean of the
12; Appendix
procedures. RI for each assay (where applicable).
F
Results for screen to confirm ratios Clarifies that a ratio of screening and
should be reported as a percent confirmatory test results for paired tests be Section 11;
correction [(screen − confirm) / screen] • reported either as a normalized ratio or a Appendix F
100. percent correction of normalized ratios.
Permits use of “indeterminate” if
consideration and interpretation of all tests Section 13;
Discourages use of “borderline” in
in LA panel does not permit a clear Appendixes
patient test report.
distinction between absence and presence of G1 and G2
LA.
*
Sections and appendixes within this column denote those within H60.
Abbreviations: APTT, activated partial thromboplastin time; dPT, dilute prothrombin time; dRVVT, dilute Russell’s viper venom
time; HPNT, hexagonal phase phospholipid neutralization test; ISTH, International Society on Thrombosis and Haemostasis;
KCT, kaolin clotting time; LA, lupus anticoagulant(s); NPP, normal pooled plasma; PL, phospholipid(s); RI, reference
interval(s); SD, standard deviation; SSC, Scientific and Standardization Committee.
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Appendix A. Algorithmic Approach to Lupus Anticoagulant Testing
Preliminary
Examination
(APTT, PT, TT)
Sample No
appropriate for LA Stop
testing
Yes
Independent System
Mixing Test
Screen
Screen
No Stop Yes
Prolonged Correction LA Not Detected
LA Not Detected (Factor Deficiency or VKAs)
(ABN) (N)
Must perform second
test system before
Yes making final decision
No (ABN)
Confirm
LA Possible (Strong LA)
LA Possible (with Factor Deficiency)
Other Inhibitor
Abnormal No (N)
delta
Yes
Factor Assays
LA REPEAT TESTING
(use three or more dilutions)
in 12 weeks
Present
Figure A1. Independent Test System*

*
Dashed blue arrows indicate lower priority attached to a mixing test.
Abbreviations: ABN, abnormal; APTT, activated partial thromboplastin time; KCT, kaolin clotting time; LA, lupus
anticoagulant(s); N, normal; PNP, platelet neutralization procedure; PT, prothrombin time; TT, thrombin time; VKA, vitamin K
antagonist(s).
Independent Assays
 Screening
– APTT or KCT
 Confirmatory
– PNP
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Number 6 H60-A
Appendix A. (Continued)
Preliminary
Examination
(APTT, PT, TT)
Sample No
testing
Yes
Paired System
Mixing Test
Screen
Screen
No Stop Yes
Prolonged Correction LA Not Detected
LA Not Detected (Factor Deficiency or VKA)
(ABN) (N)
Must perform second
test system before
Yes making final decision No (ABN)
Mixing Test Factor Assays

Confirm (use three or more dilutions)
Confirm
No
(ABN) Inhibitor Detected
(Other or Strong LA)
No (ABN)
Correction Correction
(N) (N) LA Present
Yes with Factor Deficiency
Yes
Paired Paired Paired Paired
LA REPEAT TESTING Screening Confirmatory Screen Confirm Interpretation
in 12 weeks Test Test Mixing Test Mixing Test
Present N LA not detected
ABN N LA present
ABN ABN N Factor Def/VKAs
ABN ABN ABN N LA + Factor Def
ABN ABN ABN ABN Other Inhibitor or strong LA
Figure A2. Paired Test System*

*
Dashed blue arrows indicate lower priority attached to a mixing test.
Abbreviations: ABN, abnormal; APTT, activated partial thromboplastin time; dPT, dilute prothrombin time; dRVVT, dilute
Russell’s viper venom time; Factor Def, factor deficiency; LA, lupus anticoagulant(s); N, normal; PT, prothrombin time; SCT,
silica clotting time; TT, thrombin time; VKA, vitamin K antagonist(s).
Paired Assays
 Screening and confirmatory
– dRVVT, SCT, dPT
©
Volume 34 H60-A
Appendix A. (Continued)
Preliminary
Examination
(APTT, PT, TT)
Sample No
testing
Yes
Integrated System
No Factor Assays
Positive LA Not Detected (use three or more dilutions)
(Factor Deficiency or VKAs)
Must perform second

Yes test system before
making final decision
LA
Present
REPEAT TESTING
in 12 weeks
Figure A3. Integrated Test System

Abbreviations: APTT, activated partial thromboplastin time; HPNT, hexagonal phase phospholipid neutralization test; LA, lupus
anticoagulant(s); PT, prothrombin time; TT, thrombin time; VKA, vitamin K antagonist(s).
Integrated Assay
 Screening, confirmatory, mixing test
– HPNT
©
Appendix B. Establishing Reference Intervals and Cutoff Values
Number 6
74
Lupus Anticoagulant Testing Panel: Establishment of Reference Intervals

Intrinsic Pathway Assay: Independent Tests (APTT and PNP) Common Pathway Assay: Paired Test (dRVVT) Intrinsic Pathway Assay: Integrated Test
APTT Screen PNP Confirm APTT 1:1 Mix Test dRVVT Screen dRVVT Confirm Paired Test Calculations dRVVT Screen dRVVT Confirm
HPNT
1:1 Mix Test 1:1 Mix Test
dRVVT Tube #1 Tube #2
Tube # 1 Tube #2 delta Screen Screen dRVVT Screen Screen Confirm Confirm delta
Screen Screen Screen Confirm Screen to Confirm Screen Confirm (Plasma (Plasma with
Screen (Plasma with (Plasma with (Tube #1 − 1:1 Mix 1:1 Mix Screen Confirm Screen to Confirm 1:1 Mix 1:1 Mix 1:1 Mix 1:1 Mix (Tube #1 − Tube
N Ratio 1:1 Mix N Ratio N Ratio Normalized Ratio 1:1 Mix 1:1 Mix with Hexagonal
Saline) Phospholipid) Tube #2) N Ratio ICA Normalized Ratio N Ratio ICA N Ratio ICA #2)
% Correction Saline) Phospholipid)
[Screen Patient
Normalized [Confirm 1:1
Normalized [Screen 1:1 Normalized Normalized (s) / Screen [(Screen Patient / Screen Normalized
Normalized Ratio Using [Screen 1:1 Mix (s) − dRVVT Mix (s) −
Ratio Using Mix (s) − Ratio Using Ratio Using mean of RI (s)] / RI Mean) − (Confirm Ratio Using
Ratio Using Mean RI of Screen NPP (s) / Confirm Confirm NPP
Seconds Seconds Seconds Seconds Seconds Mean RI of Screen NPP (s) Seconds Mean RI of Seconds Mean RI of [Confirm Patient (s) / Patient / Confirm RI Seconds Mean RI of Seconds Seconds Seconds
Mean RI of dRVVT Screen Patient (s)] • 1:1 Mix (s) / Confirm
APTT Screen / Screen dRVVT dRVVT Confirm mean of RI Mean) / (Screen Patient / dRVVT
APTT Screen 1:1 100 Seconds Patient (s)] •
1:1 Mix Patient (s)] • 100 Screen Confirm (s)] Screen RI Mean)] • 100 Confirm 1:1 Mix
Mix 100
Day 1
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Day 2
Sample 6
Sample 7
Sample 8
Sample 9
Sample 10
Day 3
Sample 11
Sample 12
Sample 13
Sample 14
Sample 15
Day 4
Sample 16
Sample 17
Sample 18
Sample 19
©
Sample 20
Clinical and Laboratory Standards Institute. All rights reserved.
Day 5
Sample 21
Sample 22
Sample 23
Sample 24
Sample 25
Day 6
Sample 26
Sample 27
NCCLS
Sample 28
Sample 29
Sample 30
Day 7
Sample 31
Sample 32
Sample 33
Sample 34
Sample 35
Day 8
Sample 36
Sample 37
Sample 38
Sample 39
Sample 40
Calculations
Mean
1 SD
2 SD
Cutoff (97.5%)
Max (+2 SD)
RI
Reagent 1 Lot # and Expiration date Abbreviations: APTT, activated partial thromboplastin time; dPT, dilute prothrombin time; dRVVT, dilute Russell’s viper venom time; HPNT, hexagonal phase phospholipid neutralization test; ICA, index of circulating
Reagent 2 Lot # and Expiration date anticoagulant; KCT, kaolin clotting time; LA, lupus anticoagulant; N, normalized; NPP, normal pooled plasma; PNP, platelet neutralization procedure; RI, reference interval(s); s, seconds; SCT, silica clotting time; SD, standard deviation.
Reagent 3 Lot # and Expiration date Numbers in orange cells would be automatically calculated by a spreadsheet program.
NPP Lot # and Expiration date Mean, 1 SD, and 2 SD values would be automatically calculated by a spreadsheet program.
Normal Control Lot # and Expiration date
H60-A
Abnormal Control Lot # and Expiration date This example does not suggest that only the assays noted can or should be used or that all assays represented on this table be part of an LA panel. This guideline does, however,
support the use of APTT and dRVVT as first-line screening assays. This spreadsheet can serve as a template for RI determination and shows that many values are not
determined but actually calculated, thereby minimizing the overall effort that is entailed. A list, though not exhaustive, of assay combinations that could be chosen are as
follows: SCT and dRVVT; dPT and dRVVT; KCT/PNP and dRVVT; HPNT and dRVVT.
Volume 34
©
Appendix C. Lupus Anticoagulant Testing in Relationship to Coagulation Pathways
Intrinsic Pathway Intrinsic Pathway Assays Extrinsic Pathway Assays Extrinsic Pathway
 Independent tests
Tests(screening)
(Screening)  Paired test
Test (screening/confirmatory)
(Screening/Confirmatory)
XII APTT
APTT  dPTProthrombin Time (
 dilute )
Kaolin
KCT Clotting Time (KCT)  TTI
XI  Tissue Inhibition (TTI)
 Independent test
Test(confirmatory)
(Confirmatory) VII
Platelet
 ASLA Seven LA Assay (ASLA)
 Activated
PNP Neutralization Procedure (PNP)
IX Tissue Factor
 Paired test
Test(screening/confirmatory)
VIII Silica
SCT Clotting Time (SCT)
 Integrated test
Test (screening/confirmatory/mixing)
(Screening/Confirmatory/Mixing)
  HPNT PhasePhospholipidNeutralization Test(HPNT)
Hexagonal
 Mixing tests
Tests(APTT,
(APTT,SCT,
SCT,KCT)
KCT) Intrinsic
Pathway
X Extrinsic
X
Common V Pathway Pathway V
Common Pathway
II II
Fibrinogen Fibrin Clot

Common Pathway Assays
NCCLS
 Paired tests
Tests (screening/confirmatory)
 ’s Viper Venom Time (
dilute Russell
dRVVT )
  VLVT Venom Time (VLVT)
 Mixing tests
Tests (dRVVT,
( dRVVT ,VLVT)
VLVT)
 Independent tests
Tests (screening)
(Screening)
 TextarinTime
Textarin time
  TSVT Snake Venom Time (TSVT)
 Independent Test (Confirmatory)
 EcarinTime
 Ecarin time
Intrinsic Extrinsic
Pathway Pathway
X
Common V Pathway
II
H60-A
Abbreviations: APTT, activated partial thromboplastin time; ASLA, activated seven lupus anticoagulant; dPT, diluted prothrombin time; dRVVT, dilute Russell’s viper venom
75
time; HPNT, hexagonal phase phospholipid neutralization test; KCT, kaolin clotting time; PNP, platelet neutralization procedure; SCT, silica clotting time; TSVT, Taipan snake
venom time; TTI, tissue thromboplastin inhibition; VLVT, Vipera lebetina venom time.
Number 6 H60-A
Appendix D. Diagrammatic Principles for Lupus Anticoagulant Tests
Intrinsic Pathway Assays
 Independent Lupus Anticoagulant (LA) Tests
Time for clot formation

 30 seconds
100 L CaCl2
Incubate at 37o C for ~5
5 minutes
100 L
?L Reagent (Activator + PL)
100 L
?L Plasma
Plasma
Abbreviation: PL, phospholipid(s).

Figure D1. Activated Partial Thromboplastin Time (APTT) – LA Screening Test or
Preliminary Examination Assay
SCREEN Detect clot end point

SCREEN MIX
20%
100 L CaCl2 100 L CaCl2 PPP
+
o
Incubateatat37
Incubate 37 CC for 5minutes
for ~5 minutes
LL Reagent
100 ? L
100 ? L Reagent
Kaolin Kaolin
Kaolin
=
20/80 80%
100 L
?L Plasma LL 1:4 Mix
100 ? 1:4 NPP
Abbreviations: NPP, normal pooled plasma; PPP, platelet-poor plasma.

Figure D2. Kaolin Clotting Time (KCT) – Screening Test
©
Volume 34 H60-A
Appendix D. (Continued)
LA Present Tube 1 - Tube 2 > xx delta seconds

LA Not Detected Tube 1 - Tube 2 < xx delta seconds
Detect clot end point
Add 100 L CaCl2 to both tubes

Tube #1 Tube #2
37 Co Cforfor5~5minutes
Incubate both tubes at 37 minutes
(Screen) (Confirm)
100 L APTT 100 L APTT
100 L
?LBuffer
Buffer 100 L
?LPlatelet
PlateletLysate
100 L
?L Plasma
Plasma 100L
?LPlasma
Plasma
Abbreviations: APTT, activated partial thromboplastin time; LA, lupus anticoagulant(s).
Figure D3. Platelet Neutralization Procedure – Confirmatory Test
 Paired LA Test
SCREEN Detect clot end point CONFIRM
100 L CaCl2 100 L CaCl2
Incubate at
Incubate 37o C for ~5
at 37 5 minutes
L
100 ? L Reagent 100 L
?L Reagent
Reagent
Low PL + Silica High PL + Silica
100 LL Plasma 100 LL Plasma
Abbreviation: PL, phospholipid(s).
Figure D4. Silica Clotting Time (SCT)
©
Number 6 H60-A
 Integrated LA Test
LA Present Tube 1 - Tube 2 > xx delta seconds

LA Not Detected Tube 1 - Tube 2 < xx delta seconds

Tube #1 Tube #2
37o CCfor
Incubate both tubes at 37 for5 5minutes
minutes
(Screen
(Screenand Mix)
& Mix) (Confirm
(Confirmand Mix)
& Mix)
100 L APTT 100 L APTT
50 LL NPP 50 L
?L NPP
50 LL Buffer Incubate

Incubateboth
both 50 L
?L Hexagonal
Hexagonal PL
tubes
tubesat
at37
37o CC
for
for99minutes
minutes
50 L
?L Plasma 50 L
?L Plasma
Abbreviations: APTT, activated partial thromboplastin time; LA, lupus anticoagulant(s); NPP, normal pooled plasma;
PL, phospholipid(s).
Figure D5. Hexagonal Phase Phospholipid Neutralization Test (HPNT)
Common Pathway Assays
 Paired LA Test
100 L
?L Reagent
Reagent 100 L
?L Reagent
Low PL + High PL +
dRVV + Ca ++ dRVVT
dRVV + Ca ++
Incubate at
Incubate 37o C for ~2
at 37 2 minutes
100 L
?L Plasma 100 L
?L Plasma
Abbreviations: dRVVT, dilute Russell’s viper venom time; PL, phospholipid(s).
Figure D6. dRVVT
©
Volume 34 H60-A
 Independent LA Tests
Independent Screening Assays Confirmatory Assay

Textarin Time TSVT Ecarin Time
100 L 100 L
CaCl2 Dilute Taipan
Venom + CaCl2 100 L
Dilute Ecarin
Incubate at 37 o C for ~3
3 minutes Incubate at 37 o C for ~2
2 minutes Incubate at 37 o C for ~3
3 minutes
LL
100 ? 100 ?L
L
Dilute PL + DiluteTextarin
Textarin Dilute PL
100 L
?L Plasma 100 ?
LL Plasma 200 ?L Plasma
L Plasma
Abbreviations: PL, phospholipid(s); TSVT, Taipan snake venom time.
Figure D7. Other Venom-based LA Assays
©
Number 6 H60-A
Extrinsic Pathway Assays
 Preliminary Examination Assay

 12 seconds
200 L
?L Prewarmed
Pre warmedReagent
Reagent
Thromboplastin++Ca
Ca++++
Incubate at 37o C for ~3
3 minutes
L
100 ?L Plasma
Figure D8. Prothrombin Time
 Paired LA Tests
50 L Reagent 50 L Reagent
(Activator rhTF ) (Activator rhTF)
LL Buffer
50 ? 50 L
?L PL
37oCCfor
Incubate at 37 for2
~2 minutes
minutes
100 L
?L Plasma
Plasma 100 L
?L Plasma
Plasma
Abbreviation: PL, phospholipid(s); rhTF, recombinant human tissue factor.
Figure D9. Dilute Prothrombin Time (dPT)
©
Volume 34 H60-A
Ratio 1:500
Final Ratio =
Ratio 1:50
Patient (seconds) Patient (seconds)
Ratio 1:50 = = Ratio 1:500
NPP (seconds) NPP (seconds)

37 o C for 5
Incubate both tubes at 37 ~5 minutes
Patient NPP Patient NPP
1:50 Dilution 1:500 Dilution

(“Confirmation ”) (“Screening ”)
100 L
? L 1:50 100 L
? L 1:500
Thromboplastin Thromboplastin
100 L
?L Plasma
Plasma 100 L
? L Plasma
Abbreviation: NPP, normal pooled plasma.

Figure D10. Tissue Thromboplastin Inhibition
©
Number 6 H60-A
Other Coagulation Assays
 Preliminary Examination Assay

 15 seconds
?L Prewarmed
200 L Pre -warmedReagent
Reagent
dilute Thrombin
37o CCfor
Incubate at 37 2minutes
for~2 minutes
200 L
?L Plasma
Figure D11. Thrombin Time
 Mixing Test
Perform Desired Test
100 μL + 100 μL
+ =
100% 100% 50/50
PPP NPP 1:1

Figure D12a. Mixing Test (1:1) as Applied to: Independent Screening Tests (APTT and
KCT), Paired Screening and Confirmatory Tests (SCT, dRVVT, and dPT) and as Used in
Integrated Test (HPNT)
©
Volume 34 H60-A
Perform Desired Test
160 μL + 40 μL
+ =
80% 20% 80/20
PPP NPP 4:1

Figure D12b. Mixing Test (4:1) as Applied to APTT
©
Appendix E. Table of Interferences and Limitations for Specific Lupus Anticoagulant Tests
84
Number 6
Acute Phase
Response
(Elevated
FVIII,
DTI FXa Inhibitors Fibrinogen,
Indirect Direct Platelet
FVIII FV Factor Fragments,
VKA UFH LMWH Argatroban Bivalirudin Dabigatran Hirudin Fondaparinux Rivaroxaban Inhibitors Inhibitors Deficiencies CRP)
Preliminary Examination Assays
APTT Y Y Y* Y Y Y10 Y Y26, N*11 Y1,4,9,12,14 Y Y All but VII Y17
PT Y Y* N* Y Y Y10 Y Y26 Y1,4,12,14 N Y I, II, V, VII, X N
TT N Y N* Y Y Y28 Y N N12 N N I N
Intrinsic Pathway Assays
APTT Screening Y Y Y* Y Y Y10 Y Y26, N*11 Y1,4,9,12,14 Y Y All but VII Y17
SCT Screening Y Y*7 Y Y Y Y30 Y Y, N*11 Y30 Y Y All but VII Y
* 7 30 30 8
SCT Confirmatory Y Y N Y Y Y All but VII
KCT (Screening
Y Y Y* Y Y Y Y Y Y All but VII Y
Only)
©
PNP (Confirmatory
Y3 Y21 Y3,22,23 V22
Only)
HPNT (Integrated) Y, N2,5 Y, N2,5 N*24 Y Y Y Y N15 Y3, N25 Y25, N2 Y, N* Y27
Common Pathway Assay
dRVVT Screening Y19 N* N* Y16 Y16 Y30 Y12,16 Y, N16 Y9,18,30 N25 Y25 I, II, V, X Y*
NCCLS
dRVVT
Y32 N* N* Y12, N16 Y16 Y30 Y12, N16 N Y9,15,30 N N I, II, V, X31 Y*
Confirmatory
TSVT (Screening
N13 Y6 Y29 N N9 N Y I, II
Only)
Extrinsic Pathway Assay
dPT (Screening
Y N* Y Y Y10 Y Y Y1,14 N Y I, II, V, VII, X20 N
Only)
Y = YES, listed therapeutic agents, inhibitors, factor deficiencies, and/or acute phase response will affect an LA assay; N = NO, listed therapeutic agents, inhibitors, factor deficiencies, and/or acute
NOTE phase response will not affect an LA assay; Y* = Usually YES, but may depend on the reagent or the concentration/level of interfering substance; N* = Not usually, but may depend on the reagent
or the concentration/level of the interfering substance; Blank Space = no published information available.
APTT, activated partial thromboplastin time; CRP; C reactive protein; dPT, dilute prothrombin time; dRVVT, dilute Russell’s viper venom time; DTI, direct thrombin inhibitor(s); FV, factor V;
FVIII, factor VIII; FXa, activated factor X; HPNT, hexagonal phase phospholipid test; I, fibrinogen; II, prothrombin; KCT, kaolin clotting time; LA, lupus anticoagulant(s); LMWH, low molecular
Abbreviations:
weight heparin(s), PNP, platelet neutralization procedure; PT, prothrombin time; SCT, silica clotting time; TT, thrombin time; TSVT, Taipan snake venom time; UFH, unfractionated heparin(s);
VKA, vitamin K antagonist(s).
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Volume 34 H60-A
Appendix E. (Continued)
References for Appendix E
1
Samama MM, Guinet C. Laboratory assessment of new anticoagulants. Clin Chem Lab Med.
2011;49(5):761-772.
2
Triplett DA, Barna LK, Unger GA. A hexagonal (II) phase phospholipid neutralization assay for
lupus anticoagulant identification. Thromb Haemost. 1993;70(5):787-793.
3
Brandt JT, Barna LK, Triplett DA. Laboratory identification of lupus anticoagulants: results of the
Second International Workshop for Identification of Lupus Anticoagulants: on behalf of the
Subcommittee on Lupus Anticoagulants/Antiphospholipid Antibodies of the ISTH. Thromb Haemost.
1995;74(6):1597-1603.
4
Hillarp A, Baghaei F, Fagerberg Blixter I, et al. Effects of the oral, direct factor Xa inhibitor
rivaroxaban on commonly used coagulation assays. J Thromb Haemost. 2011;9(1):133-139.
5
Triplett DA, Stocker KF, Unger GA, Barna LK. The Textarin/Ecarin Ratio: a confirmatory test for
lupus anticoagulants. Thromb Haemost. 1993;70(6):925-931.
6
Rooney AM, McNally T, Mackie IJ, Machin SJ. The Taipan snake venom time: a new test for lupus
anticoagulant. J Clin Pathol. 1994;47(6):497-501.
7
Grypiotis P, Ruffatti A, Pengo V, et al. Use of a new silica clotting time for diagnosing lupus
anticoagulant in patients who meet the clinical criteria for antiphospholipid syndrome. J Clin Lab
Anal. 2006;20(1):15-18.
8
Chantarangkul V, Tripodi A, Arbini A, Mannucci PM. Silica clotting time (SCT) as a screening and
confirmatory test for the detection of the lupus anticoagulants. Thromb Res. 1992;67(4):355-365.
9
van Os GMA, de Laat B, Kamphuisen PW, Meijers JC, de Groot PG. Detection of lupus
anticoagulant in the presence of rivaroxaban using Taipan snake venom time. J Thromb Haemost.
2011;9(8):1657-1659.
10
Lindhal TL, Baghaei F, Fagerberg Blixter I, et al.; Expert Group on Coagulation of the External
Quality Assurance in Laboratory Medicine in Sweden. Effects of the oral, direct thrombin inhibitor
dabigatran on five common coagulation assays. Thromb Haemost. 2011;105(2):371-378.
11
European Medicines Agency. Scientific discussion.
http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-
_Scientific_Discussion/human/000403/WC500027737.pdf. Accessed March 24, 2014.
12
Genzen JR, Miller JL. Presence of direct thrombin inhibitors can affect the results and interpretation
of lupus anticoagulant testing. Am J Clin Pathol. 2005;124(4):586-593.
13
Parmar K, Lefkou E, Doughty H, Connor P, Hunt BJ. The utility of the Taipan snake venom assay in
assessing lupus anticoagulant status in individuals receiving an oral vitamin K antagonist. Blood
Coagul Fibrinol. 2009;20(4):271-275.
14
Samama MM, Martinoli JL, Le Flem L, et al. Assessment of laboratory assays to measure
rivaroxaban—an oral factor Xa inhibitor. Thromb Haemost. 2010;103(4):815-825.
©
Number 6 H60-A
15
Merriman E, Kaplan Z, Butler J, Malan E, Gan E, Tran H. Rivaroxaban and false positive lupus
anticoagulant testing. Thromb Haemost. 2011;105(2):385-386.
16
Gosselin RC, King JH, Janatpur KA, Dager WH, Larkin EC, Owings JT. Effects of pentasaccharide
(fondaparinux) and direct thrombin inhibitors on coagulation testing. Arch Pathol Lab Med.
2004;128(10):1142-1145.
17
Guermazi S, Gorgi Y, Ayed K, Dellagi K. [Interference of factor VIII excess on the detection of lupus
anticoagulants by the activated partial thromboplastin time.] [Article in French.] Path Biol (Paris).
1997;45(1):28-33.
18
Cohen H, Machin SJ. Antithrombotic treatment failures in antiphospholipid syndrome: the new
anticoagulants? Lupus. 2010;19(4):486-491.
19
Olteanu H, Downes KA, Patel J, Praprotnik D, Sarode R. Warfarin does not interfere with lupus
anticoagulant detection by dilute Russell’s viper venom time. Clin Lab. 2009;55(3-4):138-142.
20
Arnout J, Vanrusselt M, Huybrechts E, Vermylen J. Optimization of the dilute prothrombin time for
the detection of lupus anticoagulant by use of a recombinant tissue thromboplastin. Br J Haematol.
1994;87(1):94-99.
21
Triplett DA, Brandt JT, Kaczor D, Schaeffer J. Laboratory diagnosis of lupus inhibitors: a
comparison of the tissue thromboplastin inhibitor procedure with a new platelet neutralization
procedure. Am J Clin Pathol. 1983;79(6):678-682.
22
Nesheim ME, Nichols WL, Cole TL, et al. Isolation and study of an acquired inhibitor of human
coagulation factor V. J Clin Invest. 1986;77(2):405-415.
23
Brandt JT, Britton A, Kraut E. A spontaneous factor V inhibitor with unexpected laboratory features.
Arch Pathol Lab Med. 1986;110(3):224-227.
24
Whitis J, Embry M, Hollensead S. Testing for lupus anticoagulants in patients on enoxaparin with
Staclot®LA [abstract]. Int J Lab Hematol. 2007;29(1):79.
25
Teruya J, West AG, Suell MN. Lupus anticoagulant assays: questions answered and to be answered.
Arch Pathol Lab Med. 2007;131(6):885-889.
26
Smogorzewska A, Brandt JT, Chandler WL, et al. Effect of fondaparinux on coagulation assays:
results of College of American Pathologists proficiency testing. Arch Pathol Lab Med.
2006;130(11):1605-1611.
27
Schouwers SM, Delanghe JR, Devreese KM. Lupus anticoagulant (LAC) testing in patients with
inflammatory status: does C-reactive protein interfere with LAC test results? Thromb Res.
2010;125(1):102-104.
28
van Ryn J, Stangier J, Haertter S, et al. Dabigatran etexilate—a novel, reversible, oral direct thrombin
inhibitor: interpretation of coagulation assays and reversal of anticoagulant activity. Thromb
Haemost. 2010;103(6):1116-1127.
29
Chen L, Rezaie AR. Proexosite-1-dependent recognition and activation of prothrombin by taipan
venom. J Biol Chem. 2004;279(17):17869-17874.
©
Volume 34 H60-A
30
Martinuzzo ME, Barrera LH, D 'adamo MA, Otaso JC, Gimenez MI, Oyhamburu J. Frequent false-
positive results of lupus anticoagulant tests in plasmas of patients receiving the new oral
anticoagulants and enoxaparin. Int J Lab Hematol. 2013 [Epub ahead of print].
31
Plumhoff EA, Schulte SJ, Elliott MA, Nichols WL, Heit JA. Sensitivity of dilute Russell’s viper
venom time (DRVVT) to coagulation factor deficiency: effect on testing for lupus anticoagulant
[Abstract #P1308, XX Congress ISTH, Sydney, Australia]. J Thromb Haemost. 2005;3 (Suppl 1).
32
Thom J, Ivey L, Gilmore G, Eikelboom JW. Evaluation of the phospholipid-rich dilute Russell’s
viper venom assay to monitor oral anticoagulation in patients with lupus anticoagulant. Blood Coagul
Fibrinolysis. 2004;15(4):353-357.
©
88
Number 6
Appendix F. Formulas and Examples for Calculating Test Results
Calculation Examples Using a Patient Sample Positive for LA
Screening Assay Confirmatory Assay

Patient 1:1 Mix Patient 1:1 Mix
Screen Screen Confirm Confirm
80 seconds 60 seconds 53 seconds 47 seconds
RI = 28 − 38 s Mix RI = 25 − 35 s RI = 26 − 34 s Mix RI = 23 − 31 s
RI Mean = 33 s RI Mean = 30 s RI Mean = 30 s RI Mean = 27 s
Normalized
Ratio or %
Test System Formulas for Calculation of Normalized Ratios or Percent Correction Calculations Using Raw Data Correction Cutoff
Independent or a Screen Patient (s) / Screen RI Mean (s) 80 / 33 2.42 < 1.09
Paired Screen
Paired Confirm b Confirm Patient (s) / Confirm RI Mean (s) 53 / 30 1.77 < 1.02
Paired Screen / c Normalized Screen to Confirm Ratio (a / b) (80 / 33) / (53 / 30) 1.37 < 1.20
Confirm [Screen Patient (s) / Screen RI Mean (s)] / [Confirm Patient (s) / Confirm RI
©
Mean (s)]
Paired Screen / d % Correction of Normalized Ratios [(a − b) / a] • 100 [(80 / 33) − (53 / 30) / (80 / 33)] • 100 27.1% < 10%
Confirm [(Screen Patient / Screen RI Mean) − (Confirm Patient / Confirm RI Mean) /
(Screen Patient / Screen RI Mean)] • 100
Independent or e ICA [(60 − 30) / 80] • 100 37.5% < 15%
Paired Screen Mix [Screen 1:1 Mix (s) − Screen 1:1 RI Mean (s) / Screen Patient (s)] • 100
Independent or f Screen 1:1 Mix (s) / Screen 1:1 RI Mean (s) 60 / 30 2.00 < 1.10
Paired Screen Mix
NCCLS
Paired Confirm g Confirm 1:1 Mix (s) / Confirm 1:1 RI Means (s) 47 / 27 1.74 < 1.20
Mix
Integrated h Tube #1 = 90 and Tube #2 = 65 90 − 65 delta = 25 s <8 s

Legend Cutoff = Values less than the cutoff indicate that LA is not detected and values greater than the cutoff indicate the presence of LA.
Abbreviations: ICA, index of circulating anticoagulant; LA, lupus anticoagulant(s); RI, reference interval(s); s, seconds.
H60-A
©
Volume 34
Appendix G1. Interpretive Comments and Rationale for Comments Based on Patient Examples (Data)
LA Testing Panel Examples
Preliminary Examination Assays Intrinsic Pathway Assay: Independent Tests (APTT and PNP) Common Pathway Assay: Paired Test (dRVVT)*
APTT Screen PNP Confirm APTT 1:1 Mix Test dRVVT Screen dRVVT Confirm Paired Test Calculations dRVVT Screen 1:1 Mix dRVVT Confirm 1:1 Mix
APTT dRVVT
Example PT PNP #1 PNP #2 APTT dRVVT dRVVT dRVVT dRVVT dRVVT
APTT APTT PNP Screen dRVVT dRVVT dRVVT dRVVT Screen to
(s) APTT TT Plasma / Plasma / Screen 1:1 Screen to Screen Screen 1:1 Confirm Confirm
Screen Screen delta (s) 1:1 Screen Screen Confirm Confirm Confirm
or (s) (s) Saline PL Mix Test Confirm 1:1 Mix Mix 1:1 Mix 1:1 Mix
(s) N Ratio (#1 − #2) Mix Test (s) N Ratio (s) N Ratio N Ratio
PT-INR (s) (s) N Ratio N Ratio (s) N Ratio (s) N Ratio
(s) % Correction
Reference Interval 10–12 30–40 9–11 31–39 < 1.20 29–37 30–38 <4 31–37 < 1.15 33–43 < 1.18 32–41 < 1.15 < 1.15 < 10% 33–40 < 1.10 33–39 < 1.10
1 LA Not Detected 11 32 10 35 1.00 ND ND ND ND ND 37 0.98 35 0.96 1.02 2.0 ND ND ND ND
2 LA Present 11 50 10 53 1.51 48 32 16 46 1.35 56 1.47 33 0.90 1.63 38.8 46 1.26 ND ND
LA Present but Assay
11 37 9 35 1.00 ND ND ND ND ND 53 1.39 34 0.93 1.49 33.1 48 1.31 ND ND
3 Dependent
LA Present With Dilution
10 40 11 41 1.17 38 33 5 36 1.06 46 1.20 35 0.96 1.25 20.0 39 1.07 ND ND
4 Effect in 1:1 Mixing Test
5 Indeterminate Results 10 39 10 41 1.18 37 35 2 35 1.03 46 1.21 41 1.12 1.08 7.4 40 1.09 ND ND
6 UFH (High Levels) 11 180 150 171 4.88 160 120 40 85 2.50 80 2.10 78 2.14 0.98 < 1.0 60 1.64 ND ND
LA Present but With UFH
11 60 75 60 1.71 65 60 5 43 1.26 46 1.20 34 0.93 1.29 22.5 43 1.18 ND ND
7 (Lower Levels)
33
42 10 41 1.17 43 41 2 35 1.03 59 1.55 55 1.51 1.03 2.6 40 1.09 37 1.03
8 VKA (INR = 3.0)
33
42 10 47 1.34 40 36 4 41 1.20 61 1.60 50 1.37 1.17 14.3 44 1.21 34 0.94
9 LA Present Despite VKA (INR = 3.0)
10 DTI 47 139 > 180 146 4.17 138 135 3 99 2.91 74 1.95 71 1.94 1.00 0.5 55 1.51 69 1.92
11 FVIII Inhibitor 12 130 9 105 3.00 98 95 3 70 2.06 34 0.89 ND ND ND ND ND ND ND ND
Mean of reference interval in seconds: APTT = 35; APTT Screen 1:1 Mix Test = 34
Mean of reference interval in seconds: dRVVT Screen = 38; dRVVT Confirm = 36.5; dRVVT Screen 1:1 Mix Test = 36.5; dRVVT Confirm 1:1 Mix Test = 36
*
Other paired test systems: intrinsic pathway assays (SCT) or extrinsic pathway (dPT)
Legend Normalized ratios for screen assays are discussed in Section 10; Normalized ratios for independent systems and normalized ratios and % corrections for paired test systems are discussed in Section 11; Normalized ratios for screen and confirm mix test
are discussed in Section 12; Appendix G2 provides suggestions for interpretive comments and rationale for those interpretive comments based upon the data presented above.
Abbreviations: APTT, activated partial thromboplastin time; DTI, direct thrombin inhibitor(s); dPT, dilute prothrombin time; dRVVT, dilute Russell’s viper venom time; FVIII, factor VIII; N, normalized; ND, not done; PL, phospholipid(s);
NCCLS
PNP, platelet neutralization procedure; PT-INR; prothrombin time-international normalized ratio; RI, reference interval(s); s, seconds; SCT, silica clotting time; TT, thrombin time; UFH, unfractionated heparin(s); VKA, vitamin K antagonist(s).
These 11 examples do not suggest that all assays, as noted, must be performed for every sample.
Testing should follow progression as suggested in Appendix A (see Figures A1, A2, and A3). All datasets are
provided in order to show how LA tests, in total, are interpreted, as shown in Appendix G2.
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Number 6
Appendix G2. Interpretive Comments and Rationale for Comments Based on Patient Examples (Comments)
Example Examples of Interpretive Comments (White Cells) and Rationale for Comments (Gray Cells)
Comment LA not detected. However, where clinical suspicion for APS is high, it may be appropriate to perform alternative LA and aPL assays.
LA Not
1 Applied assays were not able to detect LA. However, if clinical suspicion for APS is high, it may be appropriate to perform alternative LA
Detected Rationale
and aPL assays.
LA is present, as indicated by dRVVT and APTT analysis. If indicated, suggest repeat testing at or beyond 12 weeks to determine if the
Comment
LA is persistent.
2 LA Present Note that PNP and dRVVT confirmatory assays both show PL dependence: PNP delta seconds are above cutoff value and dRVVT confirm
Rationale time in seconds returned to RI. This sample is positive for LA and there is no need to perform mixing tests or factor assays to exclude
deficiencies.
LA is present, as indicated by dRVVT analysis. If indicated, suggest repeat testing at or beyond 12 weeks to determine if the LA is
Comment
LA Present but persistent.
3 Assay In the context of this reagent pairing and patient sample, only the dRVVT was positive and that is sufficient to confirm the presence of LA.
Dependent Rationale Factor assays to exclude factor deficiencies/other inhibitors are unnecessary because PT and APTT and the dRVVT confirmatory test are
all normal.
LA is present, as indicated by dRVVT and APTT analysis. If indicated, suggest repeat testing at or beyond 12 weeks to determine if the
Comment
LA is persistent.
LA Present but
Both APTT and dRVVT screening and confirmatory assays show the presence of LA (percent correction and test system ratios confirm PL
With Dilution
4 dependence) whereas mixing tests for both systems do not. The apparent lack of inhibition (false negative) is merely a manifestation of the
Effect in 1:1
Rationale dilution effect, which reduces sensitivity of mixing tests. This may be seen with weak LA wherein the normal pooled plasma in a 1:1
Mixing Test
mixing test masks the inhibitor effect. The normal PT, APTT, and TT exclude other causes of elevated clotting times, including therapeutic
©
anticoagulation.
Comment Results are indeterminate for LA. The presence of LA cannot be confirmed or excluded without repeat analysis on a new sample.
Only the APTT and dRVVT screening assays are abnormal (slightly above the upper limit of the RI). No other clotting times are elevated.
One potential explanation for the elevated APTT and dRVVT is the presence of LA because the reagents in this example are designed to be
Indeterminate
5 more responsive than routine reagents to LA. However, the confirmatory tests do not show sufficient correction for a definitive diagnosis
Results Rationale
of LA. Alternatively, a nonspecific inhibitor may be present. On the other hand, this sample may be from a normal individual whose test
results do not fall within a ± 2 SD RI. However, if the clinical suspicion for APS is high, it would be appropriate to perform alternative LA
and aPL assays.
NCCLS
Comment The prolonged APTT and TT are consistent with heparin. Heparin at this level invalidates LA testing.
The APTT reagent in this example does not contain a heparin neutralizer. The PNP result in delta seconds could be interpreted as due to
LA. However, PF4 derived from the platelets in the PNP reagent is neutralizing the UFH and generating a false-positive interpretation. The
UFH (High
6 dRVVT reagent contains a heparin neutralizer and would negate the effect of UFH. However, in this sample the level of UFH is so high
Levels) Rationale
that it exceeds the concentration of heparin (0.8–1.0 U/mL) that can be neutralized. Unless UFH concentration is known in a sample, it
cannot be assumed that the heparin has been fully quenched unless the TT test result returns to the RI subsequent to heparin neutralization
with an agent such as heparinase.
H60-A
Volume 34
Appendix G2. (Continued)
©
Example Examples of Interpretive Comments (White Cells) and Rationale for Comments (Gray Cells)
LA is present, as indicated by dRVVT analysis. If indicated, suggest repeat testing at or beyond 12 weeks to determine if the LA is
Comment
persistent.
LA Present but In this example, the dRVVT reagent contains a heparin neutralizer. The time in seconds for the dRVVT confirmatory assay has returned
7 With UFH to the RI suggesting that the heparin has been fully neutralized. Therefore, the elevated dRVVT screen results are significant and the
(Lower Levels) Rationale sample is positive for LA as indicated by the normalized screen to confirm ratio and the percent correction. The APTT reagent in this
example does not contain a heparin neutralizer. TT is prolonged, suggesting presence of heparin but result, postheparinase treatment,
returned to RI.
LA is not detected by APTT and dRVVT analysis. The PT-INR and dRVVT are consistent with the effects of VKA therapy. A weak LA
Comment
cannot be excluded. If repeat testing is performed, the patient ideally should be off anticoagulant therapy.
The effect that the VKA has on coagulation factors is shown in the results for PT-INR (factors VII, X, II) and dRVVT (factors X, II). If
INR values are greater than 1.5, these assays will be affected. The APTT (factors IX, X, II) is less affected by the deficiencies. In this
8 VKA example, the 1:1 mixing tests for APTT and dRVVT screen and confirmatory assays did show correction for the multiple acquired factor
Rationale deficiencies of VKA. This example shows how a dRVVT confirmatory 1:1 mixing test can help increase specificity. Textarin/Ecarin ratio
or TSVT/Ecarin time analysis can be useful in patients where more commonly used assays are compromised by the effect of VKA. Note
that a patient in whom LA is not detected but with an isolated factor deficiency would give similar results to the above, depending on
which factor is deficient.
LA is present, as indicated by dRVVT and APTT analysis. The PT-INR and dRVVT are also consistent with the effects of VKA therapy.
Comment
If indicated, suggest repeat testing at or beyond 12 weeks to determine if the LA is persistent.
LA Present Although the results for APTT and dRVVT on neat plasma suggest presence of LA, the results are compromised by the presence of a
9
Despite VKA coexisting abnormality (VKA) as suggested by the PT-INR. In this example, both the APTT and dRVVT screening mixing tests suggest
Rationale
an inhibitor. The dRVVT confirmatory mixing test result returned to the RI indicating that the effect from the VKA had been fully
NCCLS
corrected. Therefore, LA is present as indicated by the normalized screen to confirm ratio and the percent correction.
Comment The prolonged PT, APTT, dRVVT, and TT are consistent with a DTI. DTI invalidate LA testing.
10 DTI
Rationale DTI interfere with the final stage of in vitro coagulation (common pathway) for all these assays and LA cannot be detected.
LA is not detected. The results of the APTT and dRVVT analysis are consistent with a PL-independent inhibitor. Depending on the
Comment
clinical situation, further testing to characterize this inhibitor may be warranted.
The normal PT and TT accompanied by a markedly elevated APTT in the preliminary coagulation screen suggest a severe abnormality
11 FVIII Inhibitor
affecting the intrinsic pathway. The APTT mixing test is also elevated, confirming the presence of an inhibitor. However, the PNP result
Rationale
is normal (delta seconds less than cutoff) suggesting a PL-independent abnormality. The dRVVT, a common pathway test, is not affected
by abnormalities in the intrinsic pathway. In this example, the dRVVT screen is normal and a confirmatory assay was not performed.
Abbreviations: aPL, anti-phospholipid; APS, antiphospholipid syndrome; APTT, activated partial thromboplastin time; dRVVT, dilute Russell’s viper venom time; DTI, direct thrombin
inhibitor(s); FVIII, factor VIII; INR, international normalized ratio; LA, lupus anticoagulant(s); PF4, platelet factor 4; PL, phospholipid(s); PNP, platelet neutralization procedure; PT,
prothrombin time; PT-INR, prothrombin time-international normalized ratio; RI, reference interval(s); SD, standard deviation; TSVT, Taipan snake venom time; TT, thrombin time; UFH,
unfractionated heparin(s); VKA, vitamin K antagonist(s).
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Number 6 H60-A
The Quality Management System Approach

Clinical and Laboratory Standards Institute (CLSI) subscribes to a quality management system approach in the
development of standards and guidelines, which facilitates project management; defines a document structure via a
template; and provides a process to identify needed documents. The quality management system approach applies a
core set of “quality system essentials” (QSEs), basic to any organization, to all operations in any health care
service’s path of workflow (ie, operational aspects that define how a particular product or service is provided). The
QSEs provide the framework for delivery of any type of product or service, serving as a manager’s guide. The QSEs
are as follows:
Organization Personnel Process Management Nonconforming Event Management

Customer Focus Purchasing and Inventory Documents and Records Assessments
Facilities and Safety Equipment Information Management Continual Improvement
H60-A addresses the QSE indicated by an “X.” For a description of the other documents listed in the grid, please
refer to the Related CLSI Reference Materials section on page 94.
Event Management
Customer Focus
Nonconforming
Documents and
Purchasing and
Improvement
Facilities and
Organization
Management
Management
Assessments
Information
Equipment
Personnel
Continual
Inventory
Records
Process
Safety
X
EP05
EP06
EP09
EP12
EP15
EP24
EP28
GP41 GP41 GP41
H21
H47
H57 H57 H57
M29
©
Volume 34 H60-A
Path of Workflow
A path of workflow is the description of the necessary processes to deliver the particular product or service that the
organization or entity provides. A laboratory path of workflow consists of the sequential processes: preexamination,
examination, and postexamination and their respective sequential subprocesses. All laboratories follow these
processes to deliver the laboratory’s services, namely quality laboratory information.
H60-A addresses the clinical laboratory path of workflow processes 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
Sample management
Results review and
receipt/processing
Sample collection
Results reporting
Sample transport
and archiving
Interpretation
Examination
Examination
follow-up
ordering
Sample
X X X X X X X
GP41 GP41 GP41 GP41 GP41 GP41
H21 H21
H47 H47 H47
H57
©
Number 6 H60-A
Related CLSI Reference Materials

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.
EP06-A Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach;

Approved Guideline (2003). This document provides guidance for characterizing the linearity of a method
during a method evaluation; for checking linearity as part of routine quality assurance; and for determining
and stating a manufacturer’s claim for linear range.
EP09-A3 Measurement Procedure Comparison and Bias Estimation Using Patient Samples; Approved
Guideline—Third Edition (2013). This document addresses the design of measurement procedure
comparison experiments using patient samples and subsequent data analysis techniques used to determine the
bias between two in vitro diagnostic measurement procedures.
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
(2006). This document describes the demonstration of method precision and trueness for clinical laboratory
quantitative methods utilizing a protocol designed to be completed within five working days or less.
EP24-A2 Assessment of the Diagnostic Accuracy of Laboratory Tests Using Receiver Operating Characteristic
Curves; Approved Guideline—Second Edition (2011). This document provides a protocol for evaluating
the accuracy of a test to discriminate between two subclasses of subjects when there is some clinically relevant
reason to separate them. In addition to the use of receiver operating characteristic curves and the comparison
of two curves, the document emphasizes the importance of defining the question, selecting the sample group,
and determining the “true” clinical state.
EP28-A3c Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved
Guideline—Third Edition (2010). This document contains guidelines for determining reference values and
reference intervals for quantitative clinical laboratory tests. A CLSI-IFCC joint project.
GP41-A6 Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard—
Sixth Edition (2007). This document provides procedures for the collection of diagnostic specimens by
venipuncture, including line draws, blood culture collection, and venipuncture in children.
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.
H47-A2 One-Stage Prothrombin Time (PT) Test and Activated Partial Thromboplastin Time (APTT) Test;
Approved Guideline—Second Edition (2008). This document provides guidelines for performing the PT and
APTT tests in the clinical laboratory, for reporting results, and for identifying sources of error.
H57-A Protocol for the Evaluation, Validation, and Implementation of Coagulometers; Approved Guideline
(2008). This document provides guidance and procedures to the end user and manufacturer for the selection,
evaluation, validation, and implementation of a laboratory coagulometer.
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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COLA (MD) Association (EORLA) (Canada) Harvard Vanguard Medical Associates Japan Assn. of Clinical Reagents
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Saskatchewan (Canada) El Camino Hospital (CA) Health Canada (Canada) Jefferson Regional Medical Center (PA)
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University (AL) Emory University School of Medicine Healthscope Pathology (Australia) Jessa Ziekenhuis VZW (Belgium)
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(VA) Institute (Ethiopia) Advancement of Military Medicine- John Muir Health (CA)
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Complexe Hospitalier de la Sagamie Fayette County Memorial Hospital (OH) (CA) (MO)
(Canada) FDA Ctr. for Devices/Rad. Health Holstebro Hospital (Denmark) JPS Health Network (TX)
CompuNet Clinical Laboratories (OH) (CDRH) (MD) Holy Name Hospital (NJ) Jupiter Medical Center (FL)
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Danbury Hospital (CT) Capitulo Peninsular A.C (Mexico) Hospital Albert Einstein (Brazil) Keller Army Community Hospital (NY)
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Health (Australia) Garden City Hospital (MI) Hospital Italiano Laboratorio Central Kenora-Rainy River Reg. Lab. Program
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Delaware Public Health Laboratory (DE) George Mason University (VA) Hunt Regional Healthcare (TX) King Abdulaziz Medical City-
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Department of Veterans Affairs (DC) Golden Valley Memorial Hospital (MO) Hutt Valley Health District Health Board King Faisal Specialist Hospital &
DHHS NC State Lab of Public Health Golwilkar Metropolis (India) (New Zealand) Research Center (Saudi Arabia)
(NC) Good Samaritan Hospital (IN) IDEXX Reference Laboratories (Canada) King Hussein Cancer Center (Jordan)
Diagnostic Accreditation Program Good Samaritan Hospital Medical Center Imelda Hospital (Belgium) Kingsbrook Jewish Medical Center (NY)
(Canada) (NY) Indiana University - Chlamydia Kingston General Hospital (Canada)
Diagnostic Center for Population & Good Shepherd Medical Center (TX) Laboratory (IN) KK Women’s & Children’s Hospital
Animal Health (MI) Gottlieb Memorial Hospital (IL) Indiana University Health Bloomington (Singapore)
Diagnostic Laboratory Medicine, Inc. Grady Memorial Hospital (GA) Hospital (IN) Kuakini Health System (HI)
(MA) Grana S.A. (TX) Indiana University Health Care - Kuwait Cancer Control Center (Kuwait)
Diagnostic Laboratory Services, Inc. (HI) Grand River Hospital (Canada) Pathology Laboratory (IN) La Rabida Childrens Hospital (IL)
Diagnostic Medicine Services (Iceland) Grays Harbor Community Hospital (WA) Industrial Technology Research Institute Lab Express (AZ)
Diagnostic Services of Manitoba Great Plains Regional Med. Ctr. (NE) (ITRI) (Taiwan) Lab Médico Santa Luzia LTDA (Brazil)
(Canada) Great River Medical Center (IA) INEI-ANLIS “Dr. C. G. Malbrán” Labor Stein + Kollegen (Germany)
Dialysis Clinic, Inc. Laboratory (TN) Greater Lowell Pediatrics (MA) (Argentina) Laboratorio Bueso Arias (Honduras)
Dimensions Healthcare System Prince Greenville Memorial Medical Campus Ingalls Hospital (IL) Laboratorio Clinico Amadita P. de
George’s Hospital Center (MD) (SC) Inova Central Laboratory (VA) Gonzales S.A. (FL)
DMC University Laboratories (MI) Grey Bruce Regional Health Center Institut National de Sante Publique du Laboratorio de Referencia (FL)
Docro, Inc. (CT) (Canada) Quebec (Canada) Laboratorio Médico De Referencia
Doctor’s Data, Inc. (IL) Gritman Medical Center (ID) Institut National de Sante Publique du (Colombia)
DoctorsManagement (TN) Group Health Cooperative (WA) Quebec - INSPQ (Canada) Laboratory Alliance of Central New
Donalsonville Hospital (GA) Grove City Medical Center (PA) Institute Health Laboratories (PR) York (NY)
Door County Medical Center (WI) Guelph General Hospital (Canada) Institute of Public Health (Slovenia) Laboratory for Medical Microbiology
Dr Sulaiman Al Habib Medical Group Institute of Tropical Medicine Dept. of and Infectious Diseases (Netherlands)
(Saudi Arabia) Clinical Sciences (Belgium) Laboratory Medicin Dalarna (Sweden)
Drisc
Laboratory of Clinical Biology Med. Laboratories Duesseldorf Mt. Sinai Hospital Medical Center (IL) NSW Health Pathology (Australia)
Ziekenhuis Oost-Limburg (ZOL) (Germany) MultiCare Health Systems (WA) NTD Laboratories Inc (NY)
(Belgium) Medecin Microbiologiste (Canada) Munson Medical Center (MI) NW Physicians Lab (WA)
Laboratory of Veterinary Medicine Media Lab, Inc. (GA) Muskoka Algonquin Healthcare (Canada) Oakton Community College (IL)
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LabPlus Auckland District Health Board Medical Center Hospital (TX) Nanticoke Memorial Hospital (DE) Ochsner Clinic Foundation (LA)
(New Zealand) Medical Center of Central Georgia (GA) Nash General Hospital / Laboratory (NC) Oconee Memorial Hospital (SC)
LAC/USC Medical Center (CA) Medical Centre Ljubljana (Slovenia) Nassau County Medical Center (NY) Octapharma Plasma (NC)
Lafayette General Medical Center (LA) Medical College of Virginia Hospital National Cancer Institute, CCR, LP (MD) Odense University Hospital (Denmark)
Lahey Clinic (MA) (VA) National Cancer Institute, CDP, NIH Office of Medical Services Laboratory
Lake Charles Memorial Hospital (LA) Medical Laboratories of Windsor, LTD (MD) (DC)
Lake Norman Regional Medical Center (Canada) National Food Institute Technical Ohio Health Laboratory Services (OH)
(NC) Medical Laboratory Sciences Council of University of Denmark (Denmark) Ohio State University Hospitals (OH)
Lakeland Regional Laboratories (MI) Nigeria (Nigeria) National Health Laboratory Service C/O Ohio Valley Medical Center (WV)
Lakeland Regional Medical Center (FL) Medical University Hospital Authority F&M Import & Export Services (South Oklahoma Heart Hospital, LLC (OK)
Lakeridge Health Corporation - Oshawa (SC) Africa) Olive View-UCLA Medical Center (CA)
Site (Canada) Medical, Laboratory & Technology National Institute of Health (Thailand) Oneida Healthcare Center (NY)
Lakeview Medical Center (WI) Consultants, LLC (DC) National Institute of Health-Maputo, Ontario Medical Association Quality
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Lancaster General Hospital (PA) Medlab Ghana Ltd. (Ghana) National Institute of Standards and Service (Canada)
Landstuhl Regional Medical Center (AE) Medstar Health (DC) Technology (MD) Onze Lieve Vrouwziekenhuis (Belgium)
Lane Regional Medical Center (LA) Memorial Health System (CO) National Institutes of Health Department Orange County Community College
Lawrence and Memorial Hospitals (CT) Memorial Hermann Healthcare System of Lab Medicine (MD) (NY)
LeBonheur Children’s Hospital (TN) (TX) National Jewish Health (CO) Orange Park Medical Center (FL)
Lee Memorial Hospital (FL) Memorial Hospital at Gulfport (MS) National Pathology Accreditation Ordre Professionnel Des Technologistes
Legacy Laboratory Services (OR) Memorial Hospital of Carbondale (IL) Advisory Council (Australia) Médicaux Du Québec (Canada)
Leiden University Medical Center Memorial Hospital of Rhode Island (RI) National Society for Histotechnology, Orebro University Hospital (Sweden)
(Netherlands) Memorial Hospital of Texas County Inc. (MD) Oregon Health and Science University
LewisGale Hospital Montgomery (VA) (OK) National University Hospital (Singapore) (OR)
Lewis-Gale Medical Center (VA) Memorial Medical Center (IL) Pte Ltd (Singapore) Oregon Public Health Laboratory (OR)
Lexington Medical Center (SC) Memorial Medical Center (PA) National University of Ireland, Galway Orillia Soldiers Memorial Hospital
L’Hotel-Dieu de Quebec (Canada) Memorial Medical Center (TX) (NUIG) (Ireland) (Canada)
Licking Memorial Hospital (OH) Memorial Regional Hospital (FL) National Veterinary Institute (Sweden) Orlando Health (FL)
LifeBridge Health Sinai Hospital (MD) Memorial Sloan Kettering Cancer Center Nationwide Children’s Hospital (OH) OSF - Saint Anthony Medical Center (IL)
LifeCare Medical Center (MN) (NY) Naval Hospital Lemoore (CA) OSF St. Elizabeth Medical Center (IL)
Lithuanian Society of Laboratory Menonnite General Hospital (PR) Naval Hospital Oak Harbor (WA) OSU Veterinary Diagnostic Laboratory
Medicine (Lithuania) Mercy Franciscan Mt. Airy (OH) Naval Medical Center San Diego (CA) (OR)
Little Company of Mary Hospital (IL) Mercy Health Center (MO) NB Department of Health (Canada) OU Medical Center (OK)
Littleton Regional Healthcare (NH) Mercy Hospital (IA) Nebraska LabLine (NE) Our Lady of the Lake Regional Medical
Lodi Health (CA) Mercy Hospital (MN) Nellis Air Force Base (NV) Center/FMOL Health System (LA)
Loma Linda University Medical Center Mercy Hospital Jefferson (MO) Netlab SA (Ecuador) Our Lady’s Hospital for Sick Children
(LLUMC) (CA) Mercy Hospital of Tiffin (OH) New Brunswick Community College (Ireland)
Lompoc Valley Medical Center (CA) Mercy Hospital St. Louis (MO) (Canada) Overlake Hospital Medical Center (WA)
London Health Sciences Center (Canada) Mercy Integrated Laboratories / Mercy New Brunswick Provincial Veterinary PA Veterinary Laboratory (PA)
Long Beach Memorial Medical Center- St. Vincent (OH) Laboratory (Canada) Pacific Diagnostic Laboratories (CA)
LBMMC (CA) Mercy Medical Center (IA) New Dar Al Shifa Hospital - Kuwait Palmer Lutheran Health Center (IA)
Long Island Jewish Medical Center (NY) Mercy Medical Center (CA) (Kuwait) Palmetto Baptist Medical Center (SC)
Longmont United Hospital (CO) Mercy Medical Center (MD) New England Baptist Hospital (MA) Palmetto Health Baptist Easley (SC)
Longview Regional Medical Center (TX) Mercy Medical Center (OH) New Hampshire Public Health Labs. Palo Alto Medical Foundation (CA)
Louisiana Office of Public Health Mercy Regional Medical Center (OH) (NH) Pamela Youde Nethersole Eastern
Laboratory (LA) Meritus Medical Laboratory (MD) New Lexington Clinic (KY) Hospital (Hong Kong East Cluster)
Louisiana State University Medical Ctr. Methodist Dallas Medical Center (TX) New London Hospital (NH) (Hong Kong)
(LA) Methodist Healthcare (TN) New Medical Centre Hospital (United Park Nicollet Methodist Hospital (MN)
Lower Mainland Laboratories (Canada) Methodist Hospital (TX) Arab Emirates) Parkview Adventist Medical Center (ME)
Loyola University Medical Center (IL) Methodist Hospital Pathology (NE) New York City Department of Health Parkview Health Laboratories (IN)
Luminex Corporation (TX) Methodist Medical Center (TN) and Mental Hygiene (NY) Parkwest Medical Center (TN)
Lummi Tribal Health Center (WA) Methodist Sugarland Hospital (TX) New York Eye and Ear Infirmary (NY) Parrish Medical Center (FL)
Lutheran Hospital of Indiana Inc. (IN) MetroHealth Medical Center (OH) New York Presbyterian Hospital (NY) Pathgroup (TN)
Lynchburg General (VA) Metropolitan Hospital Center (NY) New York State Dept. of Health (NY) Pathlab (IA)
Lyndon B. Johnson General Hospital Metropolitan Medical Laboratory (IL) New York University Medical Center Pathology Associates Medical Lab. (WA)
(TX) Miami Children’s Hospital (FL) (NY) PathWest Laboratory Medicine WA
Lyster Army Health Clinic (AL) Michigan Dept. of Community Health New Zealand Blood Service (New (Australia)
MA Dept. of Public Health Laboratories (MI) Zealand) Pavia Hospital Santurce (PR)
(MA) Michigan State University (MI) Newark Beth Israel Medical Center (NJ) PeaceHealth Laboratories (OR)
Mackenzie Health (Canada) Microbial Research, Inc. (CO) Newborn Metabolic Screening Program/ Peninsula Regional Medical Center (MD)
Mafraq Hospital (United Arab Emirates) Microbiology Specialists, Inc. (TX) Alberta Health Services (Canada) Penn State Hershey Medical Center (PA)
Magnolia Regional Health Center (MS) MicroPath Laboratories (FL) Newman Regional Health (KS) Pennsylvania Dept. of Health (PA)
Main Line Clinical Laboratories, Inc. Mid America Clinical Laboratories (IN) Niagara Health System (Canada) Pennsylvania Hospital (PA)
Lankenau Hospital (PA) Mid Coast Hospital (ME) Ninewells Hospital and Medical School Peoria Tazewell Pathology Group, P.C.
Maine General Medical Center (ME) Middelheim General Hospital (Belgium) (United Kingdom [GB]) (IL)
Margaret R. Pardee Memorial Hospital Middlesex Hospital (CT) Noble’s Hospital (United Kingdom [GB]) PerkinElmer Health Sciences, Inc. (SC)
(NC) Midland Memorial Hospital (TX) NorDx - Scarborough Campus (ME) Peterborough Regional Health Centre
Maria Parham Medical Center (NC) Midwestern Regional Medical Center Norman Regional Hospital (OK) (Canada)
Marietta Memorial Hospital (OH) (IL) North Bay Regional Health Center Peterson Regional Medical Center (TX)
Marin General Hospital (CA) Mile Bluff Medical Center / Hess (Canada) PHIA Project, NER (CO)
Marion County Public Health Memorial Hospital (WI) North Carolina Baptist Hospital (NC) Phoebe Putney Memorial Hospital (GA)
Department (IN) Milford Regional Hospital (MA) North District Hospital (China) Phoenix Children’s Hospital (AZ)
Marquette General Hospital (MI) Ministry of Health - Zambia (Zambia) North Kansas City Hospital (MO) Phoenixville Hospital (PA)
Marshfield Clinic (WI) Ministry of Health and Social Welfare - North Oaks Medical Center (LA) Physicians Choice Laboratory Services
Martha Jefferson Hospital (VA) Tanzania (Tanzania) North Philadelphia Health System-St. (NC)
Martha’s Vineyard Hospital (MA) Minneapolis Community and Technical Joseph’s Hospital (PA) Physicians Laboratory & SouthEast
Martin Luther King, Jr./Drew Medical College (MN) North Shore Hospital Laboratory (New Community College (NE)
Center (CA) Minneapolis Medical Research Zealand) Physicians Preferred Laboratory (TX)
Martin Memorial Health Systems (FL) Foundation (MN) North Shore Medical Center (MA) Piedmont Atlanta Hospital (GA)
Mary Black Memorial Hospital (SC) Minnesota Department of Health (MN) North Shore-Long Island Jewish Health Piedmont Henry Hospital (GA)
Mary Greeley Medical Center (IA) MiraVista Diagnostics (IN) System Laboratories (NY) Pioneers Memorial Health Care District
Mary Hitchcock Memorial Hospital (NH) Mission Hospitals Laboratory (NC) North York General Hospital (Canada) (CA)
Mary Washington Hospital (VA) Mississippi Baptist Medical Center (MS) Northcrest Medical Center (TN) Placer County Public Health Laboratory
Massachusetts General Hospital (MA) Mississippi Public Health Lab (MS) Northeast Georgia Health System (GA) (CA)
Massasoit Community College (MA) Missouri State Public Health Laboratory Northeastern Vermont Regional Hospital Pocono Medical Center School of
Mater Health Services - Pathology (MO) (VT) Medical Technology (PA)
(Australia) Mobile Infirmary Association (AL) Northfield Hospital & Clinics (MN) Portneuf Medical Center (ID)
Maury Regional Hospital (TN) Modesto Memorial Hospital (CA) Northridge Hospital Medical Center (CA) Poudre Valley Hospital (CO)
Mayo Clinic (MN) MolecularMD Corp. (OR) Northside Hospital (GA) Prairie Lakes Hospital (SD)
Mayo Clinic Health Systems in Waycross Monadnock Community Hospital (NH) Northside Medical Center (OH) Presbyterian Hospital - Laboratory (NC)
(GA) Mongolian Agency for Standardization Northumberland Hills Hospital (Canada) Presbyterian/St. Luke’s Medical Center
Mayo Clinic Scottsdale (AZ) and Metrology (Mongolia) Northwest Arkansas Pathology (CO)
McAlester Regional Health Center (OK) Monongahela Valley Hospital (PA) Associates (AR) Preventive Medicine Foundation
McAllen Medical Center (TX) Monongalia General Hospital (WV) Northwestern Medical Center, Inc. (VT) (Taiwan)
McCullough-Hyde Memorial Hospital Montana Department of Public Health Northwestern Memorial Hospital (IL) Princess Margaret Hospital (Hong Kong)
(OH) and Human Services (MT) Norton Healthcare (KY) Proasecal LTD (Colombia)
MCG Health (GA) Montefiore Medical Center (NY) Norwalk Hospital (CT) ProMedica Laboratory (OH)
McLaren Northern Michigan (MI) Morehead Memorial Hospital (NC) Notre Dame Hospital (Canada) Prometheus Laboratories Inc. (CA)
MCN Healthcare (CO) Morristown Hamblen Hospital (TN) Nova Scotia Association of Clinical Providence Alaska Medical Center (AK)
MD Tox Laboratoires (CA) Mount Nittany Medical Center (PA) Laboratory Managers (Canada) Providence Everett Medical Center (WA)
Meadows Regional Medical Center (GA) Mt. Auburn Hospital (MA) Nova Scotia Community College Providence Hospital (AL)
Meadville Medical Center (PA) Mt. Sinai
Mt. Sinai Hospital
Hospital (Canada)
- New York (NY) (Canada) Providence St. Joseph Medical Center
Med Health Services Laboratory (PA) Novus Path Labs (India) (CA)
Providence St. Mary Medical Center SC Department of Health and St. Luke’s Hospital (MN) The Nathan S. Kline Institute (NY)
(WA) Environmental Control (SC) St. Luke’s Hospital (MO) The Naval Hospital of Jacksonville (FL)
Provista Diagnostics (AZ) Schneck Medical Center (IN) St. Luke’s Hospital (PA) The Nebraska Medical Center (NE)
Public Health Ontario (Canada) Schuyler Hospital (NY) St. Luke’s Hospital at The Vintage (TX) The Norwegian Institute of Biomedical
Puget Sound Blood Center (WA) Scientific Institute of Public Health St. Luke’s Medical Center (AZ) Science (Norway)
Pullman Regional Hospital (WA) (Belgium) St. Luke’s Regional Medical Center (ID) The Ohio State University-Vet Hospital
Queen Elizabeth Hospital (Canada) Scott & White Memorial Hospital (TX) St. Mark’s Hospital (UT) (OH)
Queen Elizabeth Hospital (China) Scripps Health (CA) St. Mary Medical Center (CA) The Permanente Medical Group, Inc.
Queensland Health Pathology Services Scuola Di Specializzaaione- University St. Mary Medical Center (PA) (CA)
(Australia) Milano Bicocca (Italy) St. Mary’s Good Samaritan (IL) The University of Texas Medical Branch
Quest - A Society for Adult Support and Seattle Cancer Care Alliance (WA) St. Mary’s Health Center (MO) (TX)
Rehabilitation (Canada) Seattle Children’s Hospital/Children’s St. Mary’s Healthcare (NY) The University of the West Indies,
Quinte Healthcare Corp. - Belleville Hospital and Regional Medical Center St. Mary’s Hospital (CO) Trinidad Campus (Trinidad and
General Site (Canada) (WA) St. Mary’s Hospital (NJ) Tobago)
Quintiles Laboratories, Ltd. (GA) Seminole Hospital District (TX) St. Mary’s Hospital (WI) The University of Tokyo (Japan)
Ramathibodi Hospital (Thailand) Sentara Healthcare (VA) St. Mary’s Medical Center (WV) Thomas Jefferson University Hospital,
Randers Regional Hospital (Denmark) Sentinel CH SpA (Italy) St. Michael’s Hospital (WI) Inc. (PA)
Range Regional Health Services (MN) Seoul National University Hospital St. Nicholas Hospital (WI) Thunder Bay Regional Health Sciences
Rapides Regional Medical Center (LA) (Korea, Republic of) St. Olavs Hospital (Norway) Centre (Canada)
RCPA Quality Assurance Programs Pty Seton Healthcare Network (TX) St. Peter’s Bender Laboratory (NY) Torrance Memorial Medical Center (CA)
Limited (Australia) Seton Medical Center (CA) St. Peter’s Hospital (MT) Touro Infirmary (LA)
Reading Hospital (PA) Shands Jacksonville (FL) St. Rita’s Medical Center (OH) Tri-Cities Laboratory (WA)
Redlands Community Hospital (CA) Shared Hospital Laboratory (Canada) St. Tammany Parish Hospital (LA) TriCore Reference Laboratories (NM)
Regina Qu’Appelle Health Region Sharon Regional Health System (PA) St. Thomas Hospital (TN) Trident Medical Center (SC)
(Canada) Sharp Health Care Laboratory Services St. Thomas-Elgin General Hospital Trillium Health Partners Credit Valley
Regional Laboratory of Public Health (CA) (Canada) Hospital (Canada)
(Netherlands) Shiel Medical Laboratory Inc. (NY) St. Vincent Hospital (NM) Trinity Health Systems (OH)
Regional Medical Laboratory, Inc. (OK) Shore Memorial Hospital (NJ) St. Vincent’s Medical Center (FL) Trinity Hospital of Augusta (GA)
Regions Hospital (MN) Shriners Hospitals for Children (OH) Stanford Hospital and Clinics (CA) Trinity Medical Center (AL)
Reid Hospital & Health Care Services Silliman Medical Center (Philippines) Stanton Territorial Health Authority Trinity Muscatine (IA)
(IN) Silverton Health (OR) (Canada) Trumbull Memorial Hospital (OH)
Renown Regional Medical Center (NV) SIMeL (Italy) Stat Veterinary Lab (CA) Tucson Medical Center (AZ)
Research Institute of Tropical Medicine Singapore General Hospital (Singapore) State of Alabama (AL) Tuen Mun Hospital, Hospital Authority
(Philippines) Singulex (CA) State of Washington Public Health Labs (Hong Kong)
Rhode Island Dept. of Health Labs (RI) Sky Lakes Medical Center (OR) (WA) Tufts Medical Center (MA)
Rhode Island Hospital (RI) Slidell Memorial Hospital (LA) State of Wyoming Public Health Tulane Medical Center Hospital & Clinic
Rice Memorial Hospital (MN) SMDC Clinical Laboratory (MN) Laboratory (WY) (LA)
Ridgeview Medical Center (MN) Sociedad Espanola de Bioquimica Statens Serum Institut (Denmark) Tulane University Health Sciences
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Riverside Health System (VA) Sociedade Brasileira de Analises Clinicas Stillwater Medical Center (OK) Twin Lakes Regional Medical Center
Riverton Memorial Hospital (WY) (Brazil) Stony Brook University Hospital (NY) (KY)
Riverview Healthcare Assoc. (MN) Sociedade Brasileira de Patologia Clinica Stormont-Vail Regional Medical Ctr. U.S. Medical Ctr. for Federal Prisoners
Riyadh Armed Forces Hospital, (Brazil) (KS) (MO)
Sulaymainia (Saudi Arabia) South Bay Hospital (FL) Sturgis Hospital (MI) U.S. Naval Hospital, Yokosuka, Japan
RMIT University (Australia) South Bend Medical Foundation (IN) Summa Health System (OH) (AP)
Robert E. Bush Naval Hospital (CA) South Bruce Grey Health Centre Sunnybrook Health Sciences Centre UC Davis Medical Center Department of
Robert Wood Johnson University (Canada) (Canada) Pathology & Laboratory Medicine
Hospital (NJ) South County Hospital (RI) Sunrise Hospital and Medical Center (CA)
Robert Wood Johnson University South Dakota State Health Laboratory (NV) UC San Diego Health System Clinical
Hospital Rahway (NJ) (SD) SUNY Downstate Medical Center (NY) Laboratories (CA)
Rochester General Hospital (NY) South Eastern Area Laboratory Services Susan B. Allen Hospital (KS) UCI Medical Center (CA)
Rockford Memorial Hospital (IL) (Australia) Susquehanna Health System (PA) UCLA Medical Center (CA)
Roger Williams Medical Center (RI) South Miami Hospital (FL) Sutter Health (CA) UCONN Health Center (CT)
Roosevelt General Hospital (NM) South Peninsula Hospital (AK) Sutter Health Sacramento Sierra Region UCSF Medical Center China Basin (CA)
Roper St. Francis Healthcare (SC) South Texas Laboratory (TX) Laboratories (CA) UMass Memorial Medical Center (MA)
Ross University School of Veterinary South West Medical Center (KS) SV Biosystems (CA) UMC of El Paso- Laboratory (TX)
Medicine (Saint Kitts and Nevis) Southeast Alabama Medical Center (AL) Swedish American Health System (IL) UMC of Southern Nevada (NV)
Roswell Park Cancer Institute (NY) SouthEast Alaska Regional Health Swedish Medical Center (CO) Umea University Hospital (Sweden)
Rouge Valley Health System (Canada) Consortium (SEARHC) (AK) Sydney South West Pathology Service UNC Hospitals (NC)
Royal Children’s Hospital (Australia) Southern Health Care Network Liverpool Hospital (Australia) Union Clinical Laboratory (Taiwan)
Royal Hobart Hospital (Australia) (Australia) Tahoe Forest Hospital (CA) United Christian Hospital (Hong Kong)
Royal Victoria Hospital (Canada) Southern Hills Medical Center (TN) Taichung Veterans General Hospital United Clinical Laboratories (IA)
Rush Copley Medical Center (IL) Southern Maryland Hospital (MD) (Taiwan) United Health Services Hospital / Wilson
Rush Health Systems (MS) Southern Pathology Services, Inc. (PR) Taiwan Society of Laboratory Medicine Hospital Lab (NY)
Rush University Medical Center (IL) Southwest General Health Center (OH) (Taiwan) United Memorial Med Center (NY)
Russellville Hospital (AL) Southwestern Regional Medical Center Tallaght Hospital (Ireland) United States Air Force School of
SA Pathology (Australia) (OK) Tampa General Hospital (FL) Aerospace Medicine / PHE (OH)
SAAD Specialist Hospital (Saudi Arabia) Sparrow Hospital (MI) Taranaki Medlab (New Zealand) United States Coast Guard (NJ)
Sacred Heart Hospital (FL) Spaulding Hospital Cambridge (MA) Tartu University Clinics (Estonia) Universidad de Guadalajara (Mexico)
Sacred Heart Hospital (WI) Speare Memorial Hospital (NH) Tataa Biocenter (Sweden) Universidade Federal Do Rio de Janeiro
Saddleback Memorial Medical Center Spectra East (NJ) Taylor Regional Hospital (KY) (Brazil)
(CA) St Elizabeth Hospital (WI) Temple Community Hospital (CA) Universitaet Zuerich (Switzerland)
Sahlgrenska Universitetssjukhuset St Rose Dominican Hospital (AZ) Temple University Hospital - Parkinson Universitair Ziekenhuis Antwerpen
(Sweden) St. Agnes Healthcare (MD) Pavilion (PA) (Belgium)
Saint Francis Hospital & Medical Center St. Anthony Hospital (OK) Tennessee Department of Health (TN) University College Hospital (Ireland)
(CT) St. Antonius Ziekenhuis (Netherlands) Tewksbury Hospital (MA) University General Hospital (TX)
Saint Francis Medical Center (IL) St. Barnabas Medical Center (NJ) Texas A & M University (TX) University Health Network Laboratory
Saint Mary’s Regional Medical Center St. Charles Medical Center-Bend (OR) Texas Children’s Hospital (TX) Medicine Program (Canada)
(NV) St. Charles Parish Hospital (LA) Texas Department of State Health University Hospital (TX)
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Virginia Mason Medical Center (WA) FAARC (MI) Jennifer Kwon (NY) Mr. Niels Wartenberg (MN)
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Virginia Regional Medical Center (MN) Steven Brown (OR) Michael LaFleur (MA) Gary Wells (TX)
Virtua - West Jersey Hospital (NJ) Vanessa Buchan (New Zealand) Grace Largado (CA) Dr. L.A. Nilika Wijeratne (Australia)
WakeMed (NC) Karen Bush (IN) Debra Larsen (TX) Matthew A Wikler (NJ)
Warren Hospital (NJ) Donald R Callihan (MD) Professor Szu-Hee Lee MD, PhD Bernadette Wildemore (GA)
Waterbury Hospital (CT) Ms. Natalie Campbell RT (Canada) (Australia) Dr. Emily S. Winn-Deen PhD (CA)
Watson Clinic (FL) Sheldon Campbell (CT) Dr. Thomas J. Lenk PhD (CA) Mr. Dennis Winsten (AZ)
Wayne Memorial Hospital (GA) Alan T. Cariski (CA) Sarah B Leppanen (CA) Ms. Sheila M. Woodcock ART, MBA,
Weber State University (UT) A. Bjoern Carle (ME) Andrew Leung (CA) FCSMLS(D) (Canada)
Weed Army Community Hospital Dr. Maria Paz Carlos DVM, PhD, MBA Jacob B Levine (NY) Ginger Wooster (WI)
Laboratory (CA) (MD) Ernst Lindhout (Netherlands) Dr. Ching Ching Wu DVM, PhD (IN)
Weeneebayko General Hospital (Canada) Eileen Carreiro-Lewandowski (MA) Kristian Linnet (Denmark) Dr. Shangwei Wu PhD (China)
Weirton Medical Center (WV) Dr. Alexis Carter MD, FCAP, FASCP Yuqing Liu (China) Stanley Wu (GA)
Wellington Regional Medical Center (GA) Philip Lively (PA) Steven York (OH)
(FL) Dr. Jose B. Casals (Denmark) Mark Loch (MN) Michelle L. Zaharik (Canada)
Wellstar Douglas Hospital Laboratory Ning Cegielski (WA) Moushumi Lodh (India) Jing Zhang (CA)
(GA) Tony Chan (China) Stefano A. Lollai (Italy) Dr. Marcia L. Zucker PhD (NJ)
Wellstar Health Systems (GA) Mintrude Charles-Young (Canada)
Pauline Cyr (Canada)
Number 6 H60-A
NOTES
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H60A - Laboratory Testing For Lupus Anticoagulant Testing Approved April 2014

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H60A - Laboratory Testing For Lupus Anticoagulant Testing Approved April 2014

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April 2014

This document provides guidance and recommendations regarding

For further information on committee participation or to submit comments, contact CLSI.

Clinical and Laboratory Standards Institute

ISBN 1-56238-959-9 (Print)

Consensus Committee on Hematology

Kathleen M. Curran, BSMT

Document Development Committee on Laboratory Testing for the Lupus Anticoagulant

Marlies Ledford-Kraemer, MBA, Philip G. de Groot, PhD Staff

John T. Brandt, MD Sandra C. Hollensead, MD Rita Selby, MBBS, FRCPC,

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

6 Validation of Assay Systems ................................................................................................... 10

Appendix A. Algorithmic Approach to Lupus Anticoagulant Testing ................................................. 71

Appendix B. Establishing Reference Intervals and Cutoff Values ....................................................... 74

Appendix C. Lupus Anticoagulant Testing in Relationship to Coagulation Pathways ........................ 75

Appendix D. Diagrammatic Principles for Lupus Anticoagulant Tests................................................ 76

Appendix F. Formulas and Examples for Calculating Test Results ..................................................... 88

Related CLSI Reference Materials ....................................................................................................... 94

Criteria for the Laboratory Diagnosis of the Lupus Anticoagulant

C. Confirmation: evidence that prolongation of the screening test(s) demonstrates phospholipid

2. Ideally, samples should not be obtained from vascular access devices.

3. Platelet count of patient-citrated platelet-poor plasma should be < 10 × 109/L.

4. Testing may be performed on fresh or properly frozen/thawed samples.

5. Routine coagulation tests, prothrombin time-international normalized ratio, activated partial

3. Solid-phase immunoassays for antibodies against phospholipid (eg, anti-cardiolipin or anti-β2

D. Mixing Test (if performed)

F. Interpretation and Reporting

Antiphospholipid syndrome, lupus anticoagulant, lupus anticoagulant confirmatory assays, lupus

Laboratory Testing for the Lupus Anticoagulant; Approved Guideline

Two types of laboratory testing are used in making a diagnosis of APS:

 Solid-phase assays for aCL and aβ2GPI

Table 1. Historical Perspective of Laboratory Tests Used for the Detection of LA

2.1 Historical Perspective of Antiphospholipid Syndrome and Relationship to Lupus

4.1 A Note on Terminology

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

Verification focuses on whether specifications of a measurement procedure can be achieved, whereas

analyte – component represented in the name of a measurable quantity (ISO 17511).60

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

measurand – quantity intended to be measured (JCGM 200:2012).59

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

measurement uncertainty//uncertainty of measurement//uncertainty – non-negative parameter

repeatability (measurement) – measurement precision under a set of repeatability conditions of

reproducibility (measurement) – measurement precision under reproducibility conditions of

4.3 Abbreviations and Acronyms

ISTH International Society on Thrombosis and Haemostasis

 Mechanical—detects physical formation of fibrin strands

6 Validation of Assay Systems

Validation of methods for LA testing should consider the following:

 Properly stored and well characterized LA samples

6.3 Establishment of Reference Intervals and Cutoff Values

6.3.1 Normalization of Lupus Anticoagulant Screening and Confirmatory Test Results

 The mean clotting time of the RI for a specific assay69,90-93

6.4 Analytical Specificity

6.5 Diagnostic Sensitivity and Diagnostic Specificity

7 Preexamination Issues (Criterion A)

7.1 Patient Selection

Defining the appropriate patient for LA testing may be predicated on:

High-probability patients have:

 Thrombosis at unusual sites

 Late pregnancy loss

Moderate-probability patients have:

 An incidental APTT prolongation

Low-probability patients have: