Laboratory criteria for antiphospholipid syndrome: communication from the SSC of the ISTH

Abstract

Currently, we use the classification criteria for antiphospholipid syndrome (APS) formulated during the consensus conference in Sapporo and revised in Sydney 1.Miyakis S. Lockshin M.D. Atsumi T. Branch D.W. Brey R.L. Cervera R. Derksen R.H. De Groot P.G. Koike T. Meroni P.L. Reber G. Shoenfeld Y. Tincani A. Vlachoyiannopoulos P.G. Krilis S.A. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).J Thromb Haemost. 2006; 4: 295-306Crossref PubMed Scopus (5123) Google Scholar. Classification criteria for definite APS are met when at least one clinical criterion (thrombosis or pregnancy morbidity) and one laboratory criterion (lupus anticoagulant (LAC), anticardiolipin antibodies (aCL) or beta2‐glycoprotein I antibodies (aβ2GPI)) are present. Positive laboratory tests should be confirmed 12 weeks after the initial testing. Recommendations published in 2009 and 2014 by the Scientific Standardisation Subcommittee (SSC) on Lupus Anticoagulant/Phospholipid Antibodies for the detection of LAC and aCL/aβ2GPI antibodies have proven useful working documents on how to perform the assays 2.Pengo V. Tripodi A. Reber G. Rand J.H. Ortel T.L. Galli M. De Groot P.G. Update of the guidelines for lupus anticoagulant detection. Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis.J Thromb Haemost. 2009; 7: 1737-40Crossref PubMed Scopus (961) Google Scholar, 3.Devreese K.M. Pierangeli S.S. de Laat B. Tripodi A. Atsumi T. Ortel T.L. Subcommittee on Lupus Anticoagulant/Phospholipid/Dependent AntibodiesSubcommittee on Lupus Anticoagulant/Phospholipid/Dependent A. Testing for antiphospholipid antibodies with solid phase assays: guidance from the SSC of the ISTH.J Thromb Haemost. 2014; 12: 792-5Crossref PubMed Scopus (146) Google Scholar. In addition, since 2006 a huge number of publications on which laboratory tests to use have been published, addressing the sensitivity and specificity of aCL and aβ2GPI, level for positivity, isotype, other antiphospholipid antibodies (aPL), etc. There is contrasting information in these studies, which may be confusing for the end user. This is a major concern of this Subcommittee, and therefore the past and present Chairs decided to summarize the consensus on laboratory criteria, with a focus on what has changed since 2006. LAC persists as a well‐established thrombotic risk factor, illustrated in many studies and often based on the meta‐analysis of Galli 4.Galli M. Luciani D. Bertolini G. Barbui T. Lupus anticoagulants are stronger risk factors for thrombosis than anticardiolipin antibodies in the antiphospholipid syndrome: a systematic review of the literature.Blood. 2003; 101: 1827-32Crossref PubMed Scopus (824) Google Scholar, 5.Devreese K.M. Antiphospholipid antibodies: evaluation of the thrombotic risk.Thromb Res. 2012; 130: S37-40Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar. However, in this meta‐analysis no distinction was made between isolated LAC and LAC associated with positivity in ELISA tests. When isolated LAC only is considered, the risk of thrombosis is low 6.Pengo V. Testa S. Martinelli I. Ghirarduzzi A. Legnani C. Gresele P. Passamonti S.M. Bison E. Denas G. Jose S.P. Banzato A. Ruffatti A. Incidence of a first thromboembolic event in carriers of isolated lupus anticoagulant.Thromb Res. 2015; 135: 46-9Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar. For LAC we rely on a combination of two phospholipid (PL)‐dependent clotting assays as no single test has sufficient sensitivity and specificity 2.Pengo V. Tripodi A. Reber G. Rand J.H. Ortel T.L. Galli M. De Groot P.G. Update of the guidelines for lupus anticoagulant detection. Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis.J Thromb Haemost. 2009; 7: 1737-40Crossref PubMed Scopus (961) Google Scholar. With all the disadvantages of LAC testing 7.Devreese K.M. Antiphospholipid antibody testing and standardization.Int J Lab Hematol. 2014; 36: 352-63Crossref PubMed Scopus (68) Google Scholar, 8.Devreese K. Hoylaerts M.F. Laboratory diagnosis of the antiphospholipid syndrome: a plethora of obstacles to overcome.Eur J Haematol. 2009; 83: 1-16Crossref PubMed Scopus (72) Google Scholar, the search for better‐performing coagulation assays in LAC continues. Functional assays that can discriminate between thrombosis‐related and non‐thrombosis‐related aPL have been described, including thrombin generation assays 9.Devreese K. Peerlinck K. Hoylaerts M.F. Thrombotic risk assessment in the antiphospholipid syndrome requires more than the quantification of lupus anticoagulants.Blood. 2010; 115: 870-8Crossref PubMed Scopus (65) Google Scholar, 10.Zuily S. Ait Aissa K. Membre A. Regnault V. Lecompte T. Wahl D. Thrombin generation in antiphospholipid syndrome.Lupus. 2012; 21: 758-60Crossref PubMed Scopus (25) Google Scholar. Thrombin generation depends on the presence of negative phospholipid surfaces, and one single test may provide more accurate information than the multitude of clotting assays required in LAC testing today. However, these assays are not robust enough to use in routine testing yet. So, the multiple‐step procedure, with screening, mixing and confirmation steps, is still applied 1.Miyakis S. Lockshin M.D. Atsumi T. Branch D.W. Brey R.L. Cervera R. Derksen R.H. De Groot P.G. Koike T. Meroni P.L. Reber G. Shoenfeld Y. Tincani A. Vlachoyiannopoulos P.G. Krilis S.A. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).J Thromb Haemost. 2006; 4: 295-306Crossref PubMed Scopus (5123) Google Scholar, 2.Pengo V. Tripodi A. Reber G. Rand J.H. Ortel T.L. Galli M. De Groot P.G. Update of the guidelines for lupus anticoagulant detection. Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis.J Thromb Haemost. 2009; 7: 1737-40Crossref PubMed Scopus (961) Google Scholar. LAC testing in patients under treatment with anticoagulation therapy may cause erroneous results, but this is beyond the scope of this paper and will be addressed in a separate SSC recommendation.Table 1Recommended laboratory testing for the antiphospholipid syndrome1. Lupus anticoagulant (LAC) present in plasma detected according to the Scientific Standardisation Subcommittee (SSC) on Lupus Anticoagulant/Phospholipid Antibodies recommendations 2.Pengo V. Tripodi A. Reber G. Rand J.H. Ortel T.L. Galli M. De Groot P.G. Update of the guidelines for lupus anticoagulant detection. Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis.J Thromb Haemost. 2009; 7: 1737-40Crossref PubMed Scopus (961) Google Scholar2. β2GPI‐dependent anticardiolipin antibodies (aCL) of IgG/IgM isotype in plasma or serum, present at higher levels (> 99th percentile of normal controls), measured by solid phase assays (ELISA or automated systems), according to the SSC on Lupus Anticoagulant/Phospholipid Antibodies recommendations 3.Devreese K.M. Pierangeli S.S. de Laat B. Tripodi A. Atsumi T. Ortel T.L. Subcommittee on Lupus Anticoagulant/Phospholipid/Dependent AntibodiesSubcommittee on Lupus Anticoagulant/Phospholipid/Dependent A. Testing for antiphospholipid antibodies with solid phase assays: guidance from the SSC of the ISTH.J Thromb Haemost. 2014; 12: 792-5Crossref PubMed Scopus (146) Google Scholar3. β2GPI‐antibodies (aβ2GPI) of IgG/IgM isotype in plasma or serum, present at higher levels (> 99th percentile), measured by solid phase assays (ELISA or automated systems), according to the SSC on Lupus Anticoagulant/Phospholipid Antibodies recommendations 3.Devreese K.M. Pierangeli S.S. de Laat B. Tripodi A. Atsumi T. Ortel T.L. Subcommittee on Lupus Anticoagulant/Phospholipid/Dependent AntibodiesSubcommittee on Lupus Anticoagulant/Phospholipid/Dependent A. Testing for antiphospholipid antibodies with solid phase assays: guidance from the SSC of the ISTH.J Thromb Haemost. 2014; 12: 792-5Crossref PubMed Scopus (146) Google Scholar4. LAC, aCL and aβ2GPI should be positive on two or more occasions at least 12 weeks apart 1.Miyakis S. Lockshin M.D. Atsumi T. Branch D.W. Brey R.L. Cervera R. Derksen R.H. De Groot P.G. Koike T. Meroni P.L. Reber G. Shoenfeld Y. Tincani A. Vlachoyiannopoulos P.G. Krilis S.A. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).J Thromb Haemost. 2006; 4: 295-306Crossref PubMed Scopus (5123) Google Scholar, 2.Pengo V. Tripodi A. Reber G. Rand J.H. Ortel T.L. Galli M. De Groot P.G. Update of the guidelines for lupus anticoagulant detection. Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis.J Thromb Haemost. 2009; 7: 1737-40Crossref PubMed Scopus (961) Google Scholar, 3.Devreese K.M. Pierangeli S.S. de Laat B. Tripodi A. Atsumi T. Ortel T.L. Subcommittee on Lupus Anticoagulant/Phospholipid/Dependent AntibodiesSubcommittee on Lupus Anticoagulant/Phospholipid/Dependent A. Testing for antiphospholipid antibodies with solid phase assays: guidance from the SSC of the ISTH.J Thromb Haemost. 2014; 12: 792-5Crossref PubMed Scopus (146) Google Scholar5. Laboratory results need to be reviewed and interpreted in a collaboration between a clinical pathologist and a clinician who is skilled at interpreting the data6. Comprehensive aPL testing (LAC, aCL, and aβ2GPI IgG and IgM) should be carried out as triple aPL‐positive patients are at high risk of thrombosis or aPL‐related pregnancy morbidity.7. Other antiphospholipid antibody tests are not recommended yet Open table in a new tab In the Sydney criteria aβ2GPI was added as an extra laboratory criterion and not as a substitute for aCL. Consequently, we recommend performing all three tests (LAC, aCL and aβ2GPI) to diagnose APS 1.Miyakis S. Lockshin M.D. Atsumi T. Branch D.W. Brey R.L. Cervera R. Derksen R.H. De Groot P.G. Koike T. Meroni P.L. Reber G. Shoenfeld Y. Tincani A. Vlachoyiannopoulos P.G. Krilis S.A. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).J Thromb Haemost. 2006; 4: 295-306Crossref PubMed Scopus (5123) Google Scholar, 2.Pengo V. Tripodi A. Reber G. Rand J.H. Ortel T.L. Galli M. De Groot P.G. Update of the guidelines for lupus anticoagulant detection. Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis.J Thromb Haemost. 2009; 7: 1737-40Crossref PubMed Scopus (961) Google Scholar, 3.Devreese K.M. Pierangeli S.S. de Laat B. Tripodi A. Atsumi T. Ortel T.L. Subcommittee on Lupus Anticoagulant/Phospholipid/Dependent AntibodiesSubcommittee on Lupus Anticoagulant/Phospholipid/Dependent A. Testing for antiphospholipid antibodies with solid phase assays: guidance from the SSC of the ISTH.J Thromb Haemost. 2014; 12: 792-5Crossref PubMed Scopus (146) Google Scholar. Traditionally, aCL and aβ2GPI are detected by ELISA 1.Miyakis S. Lockshin M.D. Atsumi T. Branch D.W. Brey R.L. Cervera R. Derksen R.H. De Groot P.G. Koike T. Meroni P.L. Reber G. Shoenfeld Y. Tincani A. Vlachoyiannopoulos P.G. Krilis S.A. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).J Thromb Haemost. 2006; 4: 295-306Crossref PubMed Scopus (5123) Google Scholar; however, recently automated platforms with variations of the solid phase (e.g. magnetic microparticles and microspheres) and various detection systems (e.g. chemiluminescense, flow cytometry and multiplex systems) have been introduced into the market 11.de Moerloose P. Reber G. Musial J. Arnout J. Analytical and clinical performance of a new, automated assay panel for the diagnosis of antiphospholipid syndrome.J Thromb Haemost. 2010; 8: 1540-6Crossref PubMed Scopus (57) Google Scholar, 12.Gutensohn K. Vossen D. Strate A. Kersten J.F. Hofbauer M. Krieger T. Automated, semi‐automated, and manual analyses of anti‐cardiolipin and anti‐beta2‐glycoprotein I antibodies in women with a history of miscarriage.Int J Lab Hematol. 2013; 35: 150-62Crossref PubMed Scopus (3) Google Scholar, 13.Persijn L. Decavele A.S. Schouwers S. Devreese K. Evaluation of a new set of automated chemiluminescense assays for anticardiolipin and anti‐beta2‐glycoprotein I antibodies in the laboratory diagnosis of the antiphospholipid syndrome.Thromb Res. 2011; 128: 565-9Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 14.Senant M. Rostane H. Fernani‐Oukil F. Hosking F. Bellery F. Courchinoux A. Tartour E. Darnige L. Dragon‐Durey M.A. Increased performances of the biological diagnosis of the antiphospholipid syndrome by the use of a multiplex assay.J Immunol Res. 2015; 2015: 983094Crossref PubMed Scopus (6) Google Scholar, 15.Tozzoli R. Villalta D. Autoantibody profiling of patients with antiphospholipid syndrome using an automated multiplexed immunoassay system.Autoimmun Rev. 2014; 13: 59-63Crossref PubMed Scopus (24) Google Scholar, 16.Van Hoecke F. Persijn L. Decavele A.S. Devreese K. Performance of two new, automated chemiluminescence assay panels for anticardiolipin and anti‐beta2‐glycoprotein I antibodies in the laboratory diagnosis of the antiphospholipid syndrome.Int J Lab Hematol. 2012; 34: 630-40Crossref PubMed Scopus (39) Google Scholar. Automated systems have the advantage that the working conditions are more harmonized, providing a strict protocol on how to perform the assay, which may reduce the inter‐laboratory variation 17.Devreese K.M. Poncet A. Lindhoff‐Last E. Musial J. de Moerloose P. Fontana P. A multicenter study to assess the reproducibility of antiphospholipid antibody results produced by an automated system.J Thromb Haemost. 2017; 15: 91-5Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar. They perform aCL and aβ2GPI IgG/IgM assays more rapidly and are less labor intensive, providing the four results at once, instead of running multiple ELISAs. Although it is not specified in the Sydney criteria that aCL should be β2GPI dependent, it is mandatory to avoid detection of non‐cofactor‐related aCL associated with infections or several drugs 1.Miyakis S. Lockshin M.D. Atsumi T. Branch D.W. Brey R.L. Cervera R. Derksen R.H. De Groot P.G. Koike T. Meroni P.L. Reber G. Shoenfeld Y. Tincani A. Vlachoyiannopoulos P.G. Krilis S.A. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).J Thromb Haemost. 2006; 4: 295-306Crossref PubMed Scopus (5123) Google Scholar, 3.Devreese K.M. Pierangeli S.S. de Laat B. Tripodi A. Atsumi T. Ortel T.L. Subcommittee on Lupus Anticoagulant/Phospholipid/Dependent AntibodiesSubcommittee on Lupus Anticoagulant/Phospholipid/Dependent A. Testing for antiphospholipid antibodies with solid phase assays: guidance from the SSC of the ISTH.J Thromb Haemost. 2014; 12: 792-5Crossref PubMed Scopus (146) Google Scholar. Specificity of aCL assays depends on the source and amount of β2GPI 18.Pierangeli S.S. Harris E.N. A protocol for determination of anticardiolipin antibodies by ELISA.Nat Protoc. 2008; 3: 840-8Crossref PubMed Scopus (89) Google Scholar, 19.Gardiner C. Hills J. Machin S.J. Cohen H. Diagnosis of antiphospholipid syndrome in routine clinical practice.Lupus. 2013; 22: 18-25Crossref PubMed Scopus (90) Google Scholar. After the discovery of β2GPI as the most important cofactor for aPL, the aβ2GPI assay was thought to be more specific and more reliable for standardization. A debate started on the role of aCL, but soon it became clear that aβ2GPI assays suffer from the same problems of standardization and that methodologically correct β2GPI‐dependent aCL assays do have diagnostic value, with identical sensitivities and specificities to aβ2GPI assays 16.Van Hoecke F. Persijn L. Decavele A.S. Devreese K. Performance of two new, automated chemiluminescence assay panels for anticardiolipin and anti‐beta2‐glycoprotein I antibodies in the laboratory diagnosis of the antiphospholipid syndrome.Int J Lab Hematol. 2012; 34: 630-40Crossref PubMed Scopus (39) Google Scholar, 20.Devreese K. Peerlinck K. Hoylaerts M.F. Diagnostic test combinations associated with thrombosis in lupus anticoagulant positive patients.Thromb Haemost. 2011; 105: 736-8Crossref PubMed Scopus (17) Google Scholar. Not surprisingly, because all these assays use β2GPI antigen, a high correlation is observed between aCL and aβ2GPI levels 13.Persijn L. Decavele A.S. Schouwers S. Devreese K. Evaluation of a new set of automated chemiluminescense assays for anticardiolipin and anti‐beta2‐glycoprotein I antibodies in the laboratory diagnosis of the antiphospholipid syndrome.Thromb Res. 2011; 128: 565-9Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 16.Van Hoecke F. Persijn L. Decavele A.S. Devreese K. Performance of two new, automated chemiluminescence assay panels for anticardiolipin and anti‐beta2‐glycoprotein I antibodies in the laboratory diagnosis of the antiphospholipid syndrome.Int J Lab Hematol. 2012; 34: 630-40Crossref PubMed Scopus (39) Google Scholar. A major problem is the high variability between the commercially available aCL and aβ2GPI assays in classifying samples as positive or negative and in antibody level. In external quality control exercises and using human monoclonal antibodies with different reactivity towards β2GPI discriminating detection of open and closed confirmation of β2GPI, differences were demonstrated in commercially available aβ2GPI IgG ELISA assays, as well as in automated systems 21.Favaloro E.J. Wheatland L. Jovanovich S. Roberts‐Thomson P. Wong R.C. Internal quality control and external quality assurance in testing for antiphospholipid antibodies: Part I–Anticardiolipin and anti‐beta2‐glycoprotein I antibodies.Semin Thromb Hemost. 2012; 38: 390-403Crossref PubMed Scopus (41) Google Scholar, 22.Pelkmans L. Kelchtermans H. de Groot P.G. Zuily S. Regnault V. Wahl D. Pengo V. de Laat B. Variability in exposure of epitope G40‐R43 of domain i in commercial anti‐beta2‐glycoprotein I IgG ELISAs.PLoS ONE. 2013; 8: e71402Crossref PubMed Scopus (32) Google Scholar, 23.Devreese K. Kelchtermans H. de Laat B. Differences in sensitivity of two automated panels for anticardiolipin and anti‐beta2glycoprotein I antibodies in the laboratory diagnosis of antiphospholipid syndrome due to the exposure of the domain I epitope of beta2glycoprotein I on the solid phase.J Thromb Haemost. 2014; 12: 55Google Scholar. The role of IgM has been discussed based on a stronger association of IgG with thrombosis compared with IgM 4.Galli M. Luciani D. Bertolini G. Barbui T. Lupus anticoagulants are stronger risk factors for thrombosis than anticardiolipin antibodies in the antiphospholipid syndrome: a systematic review of the literature.Blood. 2003; 101: 1827-32Crossref PubMed Scopus (824) Google Scholar, 24.Galli M. Borrelli G. Jacobsen E.M. Marfisi R.M. Finazzi G. Marchioli R. Wisloff F. 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More significant IgG correlations with thrombosis were confirmed; however, significant associations for IgM were also found, but with corresponding IgG. Unavailability of paired results of IgG and IgM for each separate patient hampered the evaluation of added value of IgM and the question of how many APS patients would be missed upon omission of IgM could not be answered 25.Kelchtermans H. Pelkmans L. de Laat B. Devreese K.M. IgG/IgM antiphospholipid antibodies present in the classification criteria for the antiphospholipid syndrome: a critical review of their association with thrombosis.J Thromb Haemost. 2016; 14: 1530-48Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar. We advise continued measurement of IgG and IgM; moreover, the presence of aCL and aβ2GPI of the same isotype reinforces the clinical probability of APS 26.Pengo V. Banzato A. Bison E. Bracco A. Denas G. Ruffatti A. What have we learned about antiphospholipid syndrome from patients and antiphospholipid carrier cohorts?.Semin Thromb Hemost. 2012; 38: 322-7Crossref PubMed Scopus (57) Google Scholar. The significance of IgA aCL and aβ2GPI remains controversial 1.Miyakis S. Lockshin M.D. Atsumi T. Branch D.W. Brey R.L. Cervera R. Derksen R.H. De Groot P.G. Koike T. Meroni P.L. Reber G. Shoenfeld Y. Tincani A. Vlachoyiannopoulos P.G. Krilis S.A. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).J Thromb Haemost. 2006; 4: 295-306Crossref PubMed Scopus (5123) Google Scholar, 3.Devreese K.M. Pierangeli S.S. de Laat B. Tripodi A. Atsumi T. Ortel T.L. Subcommittee on Lupus Anticoagulant/Phospholipid/Dependent AntibodiesSubcommittee on Lupus Anticoagulant/Phospholipid/Dependent A. Testing for antiphospholipid antibodies with solid phase assays: guidance from the SSC of the ISTH.J Thromb Haemost. 2014; 12: 792-5Crossref PubMed Scopus (146) Google Scholar. Future studies are needed to investigate the role of IgA in APS‐associated clinical events. Lacking an international standard, we still have to report a level derived from the calibration curve, which can differ widely between systems 7.Devreese K.M. Antiphospholipid antibody testing and standardization.Int J Lab Hematol. 2014; 36: 352-63Crossref PubMed Scopus (68) Google Scholar. Although useful for the clinician and making positive and negative results interchangeable between different systems, semiquantitative results (low‐medium‐high) are difficult to define. Each test result above the cut‐off value calculated as higher than the 99th percentile, should be regarded as positive 3.Devreese K.M. Pierangeli S.S. de Laat B. Tripodi A. Atsumi T. Ortel T.L. Subcommittee on Lupus Anticoagulant/Phospholipid/Dependent AntibodiesSubcommittee on Lupus Anticoagulant/Phospholipid/Dependent A. Testing for antiphospholipid antibodies with solid phase assays: guidance from the SSC of the ISTH.J Thromb Haemost. 2014; 12: 792-5Crossref PubMed Scopus (146) Google Scholar. However, lower levels of antibodies are observed in certain clinical settings, especially pregnancy morbidity 27.Arachchillage D.R. Machin S.J. Mackie I.J. Cohen H. Diagnosis and management of non‐criteria obstetric antiphospholipid syndrome.Thromb Haemost. 2015; 113: 13-9Crossref PubMed Scopus (61) Google Scholar, 28.Ruffatti A. Olivieri S. Tonello M. Bortolati M. Bison E. Salvan E. Facchinetti M. Pengo V. Influence of different IgG anticardiolipin antibody cut‐off values on antiphospholipid syndrome classification.J Thromb Haemost. 2008; 6: 1693-6Crossref PubMed Scopus (111) Google Scholar. The clinical relevance of aCL and/or aβ2GPI results that are below the 99th percentile needs to be studied and better defined using standardized laboratory methods. To characterize the patient's antibody profile, all three tests should be performed, preferentially in the same sample. Antibody profiles were already introduced into the Sydney criteria, categorizing patients into groups with one or more laboratory criteria (in any combination) present 1.Miyakis S. Lockshin M.D. Atsumi T. Branch D.W. Brey R.L. Cervera R. Derksen R.H. De Groot P.G. Koike T. Meroni P.L. Reber G. Shoenfeld Y. Tincani A. Vlachoyiannopoulos P.G. Krilis S.A. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).J Thromb Haemost. 2006; 4: 295-306Crossref PubMed Scopus (5123) Google Scholar. A modification of this categorization was described, taking into account the type and the number of positive tests 29.Pengo V. Biasiolo A. Pegoraro C. Cucchini U. Noventa F. Iliceto S. Antibody profiles for the diagnosis of antiphospholipid syndrome.Thromb Haemost. 2005; 93: 1147-52Crossref PubMed Scopus (256) Google Scholar. Evidence has shown that patients with more than one positive test, and particularly those patients with triple positivity (LAC and aCL and aβ2GPI; same isotype), show the strongest association with thrombotic and obstetric APS 30.Ruffatti A. Tonello M. Del Ross T. Cavazzana A. Grava C. Noventa F. Tona F. Iliceto S. Pengo V. Antibody profile and clinical course in primary antiphospholipid syndrome with pregnancy morbidity.Thromb Haemost. 2006; 96: 337-41Crossref PubMed Scopus (142) Google Scholar, 31.Pengo V. Ruffatti A. Legnani C. Gresele P. Barcellona D. Erba N. Testa S. Marongiu F. Bison E. Denas G. Banzato A. Padayattil Jose S. Iliceto S. Clinical course of high‐risk patients diagnosed with antiphospholipid syndrome.J Thromb Haemost. 2010; 8: 237-42Crossref PubMed Scopus (429) Google Scholar. Moreover, clinical studies confirm that triple positivity in APS patients and carriers indicates a high risk of recurrence of thrombosis or development of a first thrombosis, respectively 32.Pengo V. Ruffatti A. Legnani C. Testa S. Fierro T. Marongiu F. De Micheli V. Gresele P. Tonello M. Ghirarduzzi A. Bison E. Denas G. Banzato A. Padayattil Jose S. Iliceto S. Incidence of a first thromboembolic event in asymptomatic carriers of high‐risk antiphospholipid antibody profile: a multicenter prospective study.Blood. 2011; 118: 4714-8Crossref PubMed Scopus (335) Google Scholar, 33.Mustonen P. Lehtonen K.V. Javela K. Puurunen M. Persistent antiphospholipid antibody (aPL) in asymptomatic carriers as a risk factor for future thrombotic events: a nationwide prospective study.Lupus. 2014; 23: 1468-76Crossref PubMed Scopus (72) Google Scholar. Double‐positive patients (mostly LAC negative) are generally at lower risk. Probably, in these patients aβ2GPI level is insufficient to induce LAC positivity 34.Pengo V. Biasiolo A. Bison E. Chantarangkul V. Tripodi A. Italian Federation of Anticoagulation Clinics (FCSA)Antiphospholipid antibody ELISAs: survey on the performance of clinical laboratories assessed by using lyophilized affinity‐purified IgG with anticardiolipin and anti‐beta2‐Glycoprotein I activity.Thromb Res. 2007; 120: 127-33Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar. Single‐positive patients (LAC, aCL or aβ2GPI) are less likely to develop aPL‐related events 1.Miyakis S. Lockshin M.D. Atsumi T. Branch D.W. Brey R.L. Cervera R. Derksen R.H. De Groot P.G. Koike T. Meroni P.L. Reber G. Shoenfeld Y. Tincani A. Vlachoyiannopoulos P.G. Krilis S.A. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).J Thromb Haemost. 2006; 4: 295-306Crossref PubMed Scopus (5123) Google Scholar. 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In making antibody profiles, we should be aware of inter‐assay and inter‐laboratory variability and the performance characteristics of the assays: a sample assigned positive in one assay does not automatically test positive in an assay from a different manufacturer or in another laboratory 7.Devreese K.M. Antiphospholipid antibody testing and standardization.Int J Lab Hematol. 2014; 36: 352-63Crossref PubMed Scopus (68) Google Scholar, 22.Pelkmans L. Kelchtermans H. de Groot P.G. Zuily S. Regnault V. Wahl D. Pengo V. de Laat B. Variability in exposure of epitope G40‐R43 of domain i in commercial anti‐beta2‐glycoprotein I IgG ELISAs.PLoS ONE. 2013; 8: e71402Crossref PubMed Scopus (32) Google Scholar. Agreement for triple positivity between systems needs to be further studied, but looks promising 37.Iwaniec T. Kaczor M.P. Celinska‐Lowenhoff M. Polanski S. Musial J. Identification of patients with triple antiphospholipid antibody positivity is platform and method independent.Pol Arch Med Wewn. 2016; 126: 19-24PubMed Google Scholar, 38.Pengo V. Four good reasons to appreciate triple positivity.Pol Arch Med Wewn. 2016; 126: 7-8PubMed Google Scholar. Testing with other methods can be useful in patients with high clinical suspicion. Because interpretation of aPL results may be challenging, test results should always be related to clinical symptoms and an interaction between the laboratory and clinician is essential. The β2GPI domain I antibodies (aD1), a subgroup of IgG aβ2GPI, are strongly associated with thrombosis 39.de Laat B. Derksen R.H. Urbanus R.T. de Groot P.G. IgG antibodies that recognize epitope Gly40‐Arg43 in domain I of beta 2‐glycoprotein I cause LAC, and their presence correlates strongly with thrombosis.Blood. 2005; 105: 1540-5Crossref PubMed Scopus (333) Google Scholar, 40.de Laat B. Pengo V. Pabinger I. Musial J. Voskuyl A.E. 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A limited number of studies have evaluated whether the aD1 are independent risk factors for thrombosis, and showed that aD1 had no added value to the current aPL panel 42.De Craemer A.S. Musial J. Devreese K.M. Role of anti‐domain 1‐beta2 glycoprotein I antibodies in the diagnosis and risk stratification of antiphospholipid syndrome.J Thromb Haemost. 2016; 14: 1779-87Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 45.Iwaniec T. Kaczor M.P. Celinska‐Lowenhoff M. Polanski S. Musial J. Clinical significance of anti‐domain 1 beta2‐glycoprotein I antibodies in antiphospholipid syndrome.Thromb Res. 2017; 153: 90-4Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar. So far, aD1 measured by the only available commercial assay should be considered as a confirmation of the higher thrombotic risk, rather than a candidate for replacement of the aβ2GPI. Recently it was shown, using the monoclonal antibodies that detect the percentage of exposure of epitope G40‐R43 on domain I in full‐length beta2GPI, that in these commercial aD1 assays, not all epitopes on domain I are exposed. However, we cannot exclude that other epitopes on domain I may play a role. Antibodies to other domains of β2GPI are not associated with thrombotic events 46.Pengo V. Ruffatti A. Tonello M. Hoxha A. Bison E. Denas G. Padayattil Jose S. Zoppellaro G. Bracco A. Banzato A. Antibodies to Domain 4/5 (Dm4/5) of beta2‐Glycoprotein 1 (beta2GP1) in different antiphospholipid (aPL) antibody profiles.Thromb Res. 2015; 136: 161-3Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar. Antibodies to phosphatidylserine/prothrombin (aPS/PT) have been investigated in addition to the current aPL panel in APS patients with favorable results, regarding their potential diagnostic value 47.Sciascia S. Sanna G. Murru V. Roccatello D. Khamashta M.A. Bertolaccini M.L. Anti‐prothrombin (aPT) and anti‐phosphatidylserine/prothrombin (aPS/PT) antibodies and the risk of thrombosis in the antiphospholipid syndrome. A systematic review.Thromb Haemost. 2014; 111: 354-64Crossref PubMed Scopus (171) Google Scholar. Mainly associated with LAC, their additional value in APS diagnosis needs to be confirmed 48.Hoxha A. Mattia E. Tonello M. Grava C. Pengo V. Ruffatti A. Antiphosphatidylserine/prothrombin antibodies as biomarkers to identify severe primary antiphospholipid syndrome.Clin Chem Lab Med. 2017; 55: 890-8Crossref PubMed Scopus (41) Google Scholar. Although triple‐positive patients have a persistently abnormal antibody profile on follow‐up testing after 12 weeks 49.Pengo V. Ruffatti A. Del Ross T. Tonello M. Cuffaro S. Hoxha A. Banzato A. Bison E. Denas G. Bracco A. Padayattil Jose S. Confirmation of initial antiphospholipid antibody positivity depends on the antiphospholipid antibody profile.J Thromb Haemost. 2013; 11: 1527-31Crossref PubMed Scopus (83) Google Scholar, we still recommend re‐testing for confirmation after 3 months. This was originally meant to avoid over‐diagnosis by classification of transient positivity of antibodies as APS 1.Miyakis S. Lockshin M.D. Atsumi T. Branch D.W. Brey R.L. Cervera R. Derksen R.H. De Groot P.G. Koike T. Meroni P.L. Reber G. Shoenfeld Y. Tincani A. Vlachoyiannopoulos P.G. Krilis S.A. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).J Thromb Haemost. 2006; 4: 295-306Crossref PubMed Scopus (5123) Google Scholar, 2.Pengo V. Tripodi A. Reber G. Rand J.H. Ortel T.L. Galli M. De Groot P.G. Update of the guidelines for lupus anticoagulant detection. Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis.J Thromb Haemost. 2009; 7: 1737-40Crossref PubMed Scopus (961) Google Scholar. However, reproducing the same test results as for the initial positive test after 3 months ensures the reliability of the positive test, which is important in the context of poor standardization and interferences with effect on the test result 7.Devreese K.M. Antiphospholipid antibody testing and standardization.Int J Lab Hematol. 2014; 36: 352-63Crossref PubMed Scopus (68) Google Scholar, 49.Pengo V. Ruffatti A. Del Ross T. Tonello M. Cuffaro S. Hoxha A. Banzato A. Bison E. Denas G. Bracco A. Padayattil Jose S. Confirmation of initial antiphospholipid antibody positivity depends on the antiphospholipid antibody profile.J Thromb Haemost. 2013; 11: 1527-31Crossref PubMed Scopus (83) Google Scholar. In conclusion, the joint efforts of independent organizations are contributing to an improved standardization of the assays and diagnosis of APS. The SSC on Lupus Anticoagulant/Phospholipid Antibodies continues to work on improvement of the detection of aPL. K. M. J. Devreese initiated and wrote the manuscript. T. L. Ortel, V. Pengo and B. de Laat encouraged the idea, critically revised the manuscript and added intellectual content. K. M. J Devreese, T. L. Ortel, V. Pengo and B. De Laat participated in discussions leading to the development of the recommendations. T. L. Ortel reports grants from Siemens and Stago, outside the submitted work. The other authors state that they have no conflict of interest. The authors want to thank all current SSC co‐chairs (C. Belizna, H. Cohen, D. Erkan and D. Wahl) for their input and comments.