Lupus anticoagulants (LAs) are a heterogeneous group of autoantibodies that interfere with in vitro phospholipid-dependent coagulation tests, resulting in abnormally prolonged clotting times due to inhibition. The antibodies are nonspecific in that they are not directed to phospholipid nor are they inhibitory for a specific coagulation factor. Instead, LA antibodies typically target neo-antigenic epitopes of certain phospholipid-binding plasma proteins, such as beta-2 glycoprotein I or prothrombin (coagulation factor II). LA can occur spontaneously and in individuals with autoimmune disorders (eg, systemic lupus erythematosus [SLE]), certain infections or inflammatory disorders, and malignancies. Some medications can also lead to LA occurrence.1
Because of the involvement of phospholipids, LAs are usually grouped in the antiphospholipid antibody family.1-3 These antibodies have clinical significance because of their association with thrombosis, recurrent fetal loss, neurological problems, and cutaneous manifestations.1,4 Accurate diagnosis is important considering the potential use of long-term anticoagulant therapy because of the high risk of recurrent thrombosis.1,3,4
The exact cause of LA is unclear and likely multifactoral. These autoantibodies can be associated with autoimmune disease (mostly SLE), chronic infections and acute or chronic inflammatory conditions, or may be related to medication use.1,4 Patients with HIV infection also have a high incidence of LA at some time in the course of their disease. These antibodies can also be found in asymptomatic elderly individuals.1,5 The most prevalent drugs implicated in drug-induced LA are procainamide, hydralazine, quinine/quinidine, isoniazid, and antipsychotic agents, although the list is expanding.6 The majority of patients with drug-induced LA have no systemic autoimmune disease or any other underlying disorder and often may have no clinical manifestations, however these drugs can induce SLE.
Guidelines and Recommendations for Laboratory Testing
Over the years, various expert groups have published LA testing guidelines that mainly outlined current expert opinion on best practice. However, gold standards and rigorous studies continue to be elusive and thus variability of recommendations exists even among experts.
The International Society on Thrombosis and Hemostasis (ISTH)-published LA testing guidelines in 1983, 1991, 1995, and 2009.7 The British Committee for Standards in Haematology (BCSH) published an update of their previous guidelines (1991 and 2000) in early 2012.8 In 2002, the College of American Pathologists (CAP) and Douglas Triplett, MD published recommendations on the collection of specimens and laboratory testing for LA.9 The Clinical and Laboratory Standards Institute (CLSI) published its first LA guideline document in April 2014.10 While there are some differences in the published recommendations, most agree on the following diagnostic laboratory criteria and recommendations.11
- Prolongation of a phospholipid-dependent clotting time
- Dilute Russell’s viper venom time (DRVVT)
- Activated partial thromboplastin time (APTT)
- Two or more tests with different assay principles should be performed for screening
- Inhibition of the prolongation must be demonstrated via a mixing test
- Mixing of patient plasma with normal plasma does not correct the prolonged clotting time
- Phospholipid dependence of the prolongation and inhibition must be demonstrated
- DRVVT confirmatory test
- APTT-based hexagonal phase phospholipid neutralization test
- APTT-based platelet neutralization procedure
- Exclude/ evaluate for confounding coagulopathies
- Factor deficiencies and inhibitors
- Direct oral anticoagulants
- Recommend testing for serum antiphospholipid (aPL) antibodies (IgG and IgM isotypes)
- Cardiolipin antibodies
- Beta-2-glycoprotein I antibodies
Recent Guidelines and Recommendations
ISTH Scientific Standardization Committee (SSC) Guidelines 2009
The most recent information and recommendations from the ISTH SSC come from the 2009 update.6 In this publication, the committee recommends the following in addition to the already aforementioned established guidelines.
- Patient selection
- Minimize inappropriate requests for LA testing
- Testing should be limited to:
- Patients who have a significant probability of having antiphospholipid syndrome (APS)
- Patients who have an unexplained prolonged APTT
- Recommendations for optimal laboratory detection of LA
- Specimen procurement and processing must be optimized
- Issues related to testing
- Screening – 2 tests of different principles (LA-responsive APTT and dRVVT reagents recommended)
- Mixing test
- Confirmatory test
- Expression of results using normalized ratios
- Interpretation of test results
- Use locally derived cutoff values (>99th percentile)
- Consider interferences such as vitamin K antagonists or heparin
- Reporting of results
- Report quantitative results with interpretation: LA positive, negative, or indeterminate
- Correlate LA testing with other aPL testing such as serologic testing for IgG and IgM anticardiolipin and/or anti-beta-2 glycoprotein I antibodies
British Committee for Standards in Haematology (BCSH) 2012
The published guidelines on the investigation and management of APS expand on the 2000 guidelines with the following additional statements and recommendations.7
- Recommend the use of the DRVVT plus 1 additional LA test (APTT or dilute prothrombin time)
- Suggest that mixing study may obscure weak LA
- State that confirmatory test is essential to demonstrate phospholipid dependence
- Caution about LA test specificity in the presence of vitamin K or heparin anticoagulant therapy
- Suggest antiphospholipid immunoassay tests
- Recommend stratifying selection of patients for testing
- Recommend repeat testing to demonstrate persistent positivity (≥12 weeks)
The Clinical and Laboratory Standards Institute (CLSI) 2014
In April 2009, a proposal was submitted to create a guideline document regarding recommendations for the diagnosis of LA with the goals to continue to build upon previous global initiatives and to harmonize with and add clarity to current guidelines. In addition, the committee worked to present information in a succinct, practical, and easy to understand format.10
The scope of the project included the following:
- Provide recommendations for performance and interpretation of screening assays, mixing tests, and confirmatory assays
- Address preexamination issues, examination concerns, and postexamination matters that pertain to interpretation of individual tests or combinations of assays
- The intended users are laboratory personnel responsible for performing LA testing, physicians (hematologists, pathologists, rheumatologists, others), external quality assurance (EQA) programs, and manufacturers of reagents used in LA testing
- Two methodologies are used for the diagnosis of APS; however, the guideline is limited to clot-based coagulation assays used as surrogates for identifying LA
The final CLSI H60-A document was released April 4, 2014, and includes recommendations about specimen collection and handling, descriptions and limitations of screening, confirmatory assays and mixing tests, determination of cutoff values and calculations associated with the various assays, and interpretation of test results.
In addition, this new document introduces a key shift in the order in which LA tests are performed. The CLSI document suggests placing less importance on the mixing study, placing it last in the sequence of testing. The mixing study is affected by several interferences and has the potential to produce false-negative or false-positive results.10 To make the laboratory diagnosis of LA, the following criteria were defined by the committee.(Used with permission. Clinical and Laboratory Standards Institute 2014)
Procurement: adherence to standardized protocols for collection and processing of blood to be used for testing
Screening: prolongation of at least 1 of 2 different phospholipid-dependent clotting assays based on different principles and coagulation pathways
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
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
Exclusion: distinguish LA from other causes of prolonged clotting times that may mask, mimic, or coexist with LA, such as anticoagulant therapies or other coagulopathies
Interpretation and Reporting: numerical results of all testing should be reported, and interpretive comments that address and integrate these results should be provided
In addition to these basic criteria, the committee also made several recommendations specific to each criterion for the laboratory diagnosis of the lupus anticoagulant.(Used with permission. Clinical and Laboratory Standards Institute, 2014)
- Testing should preferably be performed in the absence of anticoagulant therapy (except for antiplatelet therapy).
- Ideally, samples should not be obtained from vascular access devices.
- Platelet count of patient-citrated platelet-poor plasma should be <10 x 109/L.
- Testing may be performed on fresh or properly frozen/thawed samples.
- 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.
- Lupus anticoagulant-responsive activated partial thromboplastin time and dilute Russell’s viper venom time tests are recommended as the preferred minimal screening assays.
- Other tests for the lupus anticoagulant referenced in this document may supplement the preferred minimal screening tests.
- Where test design permits, results should be calculated using the mean of the reference interval and reported as a normalized ratio.
- Routine coagulation tests, prothrombin time, 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 LA testing and interpretation.
- Confirmatory assays should use the same assay principle as the abnormal screening test (eg, dRVVT and dRVVT confirm).
- 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.
- Solid phase immunoassays for antibodies to phospholipid (eg, anticardolipin or anti-β2 glycoprotein I) should not be considered as lupus anticoagulant confirmatory procedures.
Mixing Test (if performed):
- The platelet count of the normal pooled plasma should be <10 x 109/L.
- A mix ratio of one part plasma sample to one part normal pooled plasma is recommended as the preferred ratio for a mixing test.
- A mix ratio of one part plasma sample to one part normal pooled plasma is recommended as the preferred ratio for a mixing test.
- The dilution effect of a 1:1 mixing test may mask lupus anticoagulant inhibitory activity. Other mix ratios (eg, 4:1 patient plasma:normal pooled plasma) may be used, if validated by the laboratory.
- 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.
- 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).
- The lupus anticoagulant should be distinguished from anticoagulant therapies and/or other coagulation disorders which may interfere with lupus anticoagulant testing and interpretation.
- If possible, perform factor assays whenever there is suspicion of a specific factor deficiency or inhibitor, using 3 or more dilutions of patient plasma and an activated partial thromboplastin time reagent that is unresponsive to the lupus anticoagulant.
Interpretation and Reporting:
- Numerical results of all testing should be reported with reference interval or cutoff values.
- Interpretive comments that address and integrate all test results (the lupus anticoagulant panel) should be provided.
- The interpretive report should indicate whether lupus anticoagulant is present, not detected, or indeterminate.
- Solid-phase assays for antibodies against cardiolipin and/or anti-β2 glycoprotein I are recommended as part of an evaluation for antiphospholipid syndrome.
- If lupus anticoagulant is present, the test panel should be repeated at or beyond 12 weeks to determine persistence of lupus anticoagulant as part of the evaluation for antiphospholipid syndrome.
The 4 cardinal criteria for LA assessment and diagnosis – Screen, Confirm, Mix, Exclusion – are paramount for guiding accurate testing and interpretive reporting as reconfirmed in recently updated LA testing guidelines: ISTH-SSC (2009); BCSH (2012); CLSI H60-A (2014).
Adherence to specific recommendations, insofar as feasible and practical, contributes importantly to the diagnostic specificity of LA testing. Although recent guidelines will assist laboratories in the accurate detection of LA, there is need for additional improvement in recommendations and guidelines in LA testing.
Authored by William L Nichols MD and Elizabeth Plumhoff
- Nichols WL, Kottke-Marchant K, Ledford-Kraemer MR, Homburger HA, Cardel LA: Lupus anticoagulants, antiphospholipid antibodies, and antiphospholipid syndrome. Chapter 39 In Laboratory Hematology Practice. Edited by K Kottke-Marchant and BH Davis. Oxford, UK, Wiley-Blackwell, 2012, pp 509–525
- Brandt JT, Barna LK, Triplett DA: Laboratory identification of lupus anticoagulants: results of the second international workshop for identification of lupus anticoagulants. Thromb Haemost 1995;74:1597–1603
- Levine JS, Branch DW, Rauch J: The antiphospholipid syndrome. N Engl J Med 2002;346:752–763
- Bontempo FA: The Lupus Anticoagulant – An Update. The Institute for Transfusion Medicine. Transfusion Medicine Updates. May 2001
- Petri M: Update on anti-phospholipid antibodies in SLE: the Hopkins’ Lupus Cohort. Lupus 2012;19(4):419-423
- Dlott JS, Roubey RA: Drug-induced lupus anticoagulants and antiphospholipid antibodies. Curr Rheumatol Rep 2012 Feb;14(1):71-78
- Pengo V, Tripodi A, Reber G, et al: Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis. Update of the guidelines for lupus anticoagulant detection. J Thromb Haemost 2009;7(10):1737–1740
- Keeling D, Mackie I, Moore GW, et al: British Committee for Standards in Haematology. Guidelines on the investigation and management of antiphospholipid syndrome. Br J Haematol 2012;157(1):47–58
- Triplett DA: Antiphospholipid antibodies. Arch Pathol Lab Med. 2002 Nov;126(11):1424–1429
- Clinical and Laboratory Standards Institute (CLSI). Laboratory Testing for the Lupus Anticoagulant; Approved Guideline. CLSI document H60-A. Wayne, PA, Clinical Laboratory Standards Institute, 2014
- Moore GW: Recent guidelines and recommendations for laboratory detection of lupus anticoagulants. Semin Thromb Hemost 2014;40:163–171