Antiphospholipid syndrome (APS) is a hypercoagulation condition associated with the incidence of heterogenic antiphospholipid antibodies (aPLs), which non-specifically affect hemostasis processes. APS is clinically manifested by recurrent arterial and venous thromboses and reproduction losses. The aPL antibodies, which may induce clinical manifestations of APS, include criteria antibodies anti-cardiolipin, anti-β2-glycoprotein-I, and lupus anticoagulant, but also non-criteria antibodies, for example anti-β2-glycoprotein-I domain I, anti-phosphatidylserine/prothrombin, anti-annexin V, and many others. APS occurs mostly in patients of younger and middle age, most frequently in females. Laboratory diagnostics of APS are quite difficult, as they include a wide spectrum of examining methods, which are based on various principles of detection and are performed using various laboratory techniques. The objective of the review is to describe the current state of potentially examined biomarkers and methods in APS diagnostics. The aforementioned biomarkers are lupus anticoagulant, anti-β2-glycoprotein-I, anti-cardiolipin, anti-β2-glycoprotein-I domain I, anti-phosphatidylserine/prothrombin, anti-β2-glycoprotein-I IgA, anti-cardiolipin IgA, anti-annexin V and II, anti-prothrombin, anti-cardiolipin/vimentin, anti-protein S/protein C, and antibodies against phospholipid antigens for whose diagnostics we may use some of the methods established for a long time and some of the modern methods-the coagulation method for the determination of lupus anticoagulant (LA), enzyme-linked imunosorbent assay (ELISA), chemiluminescence analysis (CLIA), multiplex fluorescence flow immunoassay (MFFIA), fluorescence enzyme immunoassay (EliA), line immunoassay (LIA), multiline dot assay (MLDA), and thin-layer chromatography (TLC). Conclusion: Antibodies against phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, cardiolipin/vimentin complex, and annexin V are currently the most studied new markers. However, these assays have not been standardized until now, both from the laboratory and clinical point of view. In this review we summarize the evidence of the most studied aPL markers and their potential clinical significance in seronegative APS (SN-APS).

Department of Hematology Oncology Faculty of Medicine and Dentistry University Hospital Olomouc Palacky University Olomouc 77900 Olomouc Czech Republic

Department of Immunology Faculty of Medicine and Dentistry Palacky University Olomouc and Faculty Hospital 775 15 Olomouc Czech Republic

Department of Internal Medicine 3 Nephrology Rheumatology and Endocrinology University Hospital Olomouc and Faculty of Medicine and Dentistry Palacky University Olomouc 77900 Olomouc Czech Republic

Masaryk Hospital Usti nad Labem Department Clinical Hematology 40113 Usti nad Labem Czech Republic

See more in PubMed

Hughes G.R. Thrombosis, abortion, cerebral disease, and the lupus anticoagulant. Br. Med. J. 1983;287:1088–1089. doi: 10.1136/bmj.287.6399.1088. PubMed DOI PMC

López-Pedrera C., Barbarroja N., Patiño-Trives A.M., Collantes E., Aguirre M.A., Perez-Sanchez C. New Biomarkers for Atherothrombosis in Antiphospholipid Syndrome: Genomics and Epigenetics Approaches. Front. Immunol. 2019;10:764. doi: 10.3389/fimmu.2019.00764. PubMed DOI PMC

Radic M., Pattanaik D. Cellular and Molecular Mechanisms of Anti-Phospholipid Syndrome. Front. Immunol. 2018;9:969. doi: 10.3389/fimmu.2018.00969. PubMed DOI PMC

Schreiber K., Sciascia S., de Groot P.G., Devreese K., Jacobsen S., Ruiz-Irastorza G., Salmon J.E., Shoenfeld Y., Shovman O., Hunt B.J. Antiphospholipid syndrome. Nat. Rev. Dis. Primers. 2018;4:17103. doi: 10.1038/nrdp.2017.103. PubMed DOI

Abeysekera R.A., Wazil A.W.M., Nanayakkara N., Ratnatunga N.V.I., Fernando K.M., Thinnarachchi J. Primary antiphospholipid syndrome presenting as antiphospholipid syndrome nephropathy: A case report. J. Med. Case Rep. 2015;9:28. doi: 10.1186/1752-1947-9-28. PubMed DOI PMC

Rand J.H. The antiphospholipid syndrome. Hematol. Am. Soc. Hematol. Educ. Program. 2007;2007:136–142. doi: 10.1182/asheducation-2007.1.136. PubMed DOI

Meroni P.L., Toubi E., Shoenfeld Y. Are Anti-Phospholipid Syndrome and Systemic Lupus Erythematosus Two Different Diseases? A 10-Year Late Remake. Isr. Med. Assoc. J. 2019;21:491–493. PubMed

Cervera R. Antiphospholipid syndrome. Thromb. Res. 2017;151:S43–S47. doi: 10.1016/S0049-3848(17)30066-X. PubMed DOI

Cervera R., Serrano R., Pons-Estel G.J., Ceberio-Hualde L., Shoenfeld Y., de Ramón E., Buonaiuto V., Jacobsen S., Zeher M.M., Tarr T., et al. Morbidity and mortality in the antiphospholipid syndrome during a 10-year period: A multicentre pro-spective study of 1000 patients. Ann. Rheum. Dis. 2015;74:1011–1018. doi: 10.1136/annrheumdis-2013-204838. PubMed DOI

Garcia D., Erkan D. Diagnosis and Management of the Antiphospholipid Syndrome. N. Engl. J. Med. 2018;378:2010–2021. doi: 10.1056/NEJMra1705454. PubMed DOI

Rodríguez-Pintó I., Moitinho M., Santacreu I., Shoenfeld Y., Erkan D., Espinosa G., Cervera R. Catastrophic antiphospholipid syndrome (CAPS): Descriptive analysis of 500 patients from the International CAPS Registry. Autoimmun. Rev. 2016;15:1120–1124. doi: 10.1016/j.autrev.2016.09.010. PubMed DOI

Liu L., Sun D. Pregnancy outcomes in patients with primary antiphospholipid syndrome: A systematic review and meta-analysis. Medicine. 2019;98:e15733. doi: 10.1097/MD.0000000000015733. PubMed DOI PMC

Whitaker K.L. Antiphospholipid antibody syndrome: The difficulties of diagnosis. JAAPA. 2017;30:10–14. doi: 10.1097/01.JAA.0000526771.67820.59. PubMed DOI

Khamashta M.A. Management of thrombosis and pregnancy loss in the antiphospholipid syndrome. Lupus. 1998;7:S162–S165. doi: 10.1177/096120339800700235. PubMed DOI

Ruiz-Irastorza G., Crowther M., Branch W., Khamashta M.A. Antiphospholipid syndrome. Lancet. 2010;376:1498–1509. doi: 10.1016/S0140-6736(10)60709-X. PubMed DOI

Miyakis S., Lockshin M.D., Atsumi T., Branch D.W., Brey R.L., Cervera R., Derksen R.H.W.M., De Groot P.G., Koike T., Meroni P.L., et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS) J. Thromb. Haemost. 2006;4:295–306. doi: 10.1111/j.1538-7836.2006.01753.x. PubMed DOI

Devreese K.M.J., Ortel T.L., Pengo V., De Laat B., Antibodies T.S.O.L.A. Laboratory criteria for antiphospholipid syndrome: Communication from the SSC of the ISTH. J. Thromb. Haemost. 2018;16:809–813. doi: 10.1111/jth.13976. PubMed DOI

Pengo V., Banzato A., Bison E., Bracco A., Denas G., Ruffatti A. What have we learned about antiphospholipid syndrome from patients and antiphospholipid carrier co-horts? Semin. Thromb. Hemost. 2012;38:322–327. PubMed

Pengo V., Ruffatti A., Del Ross T., Tonello M., Cuffaro S., Hoxha A., Banzato A., Bison E., Denas G., Bracco A., et al. Confirmation of initial antiphospholipid antibody positivity depends on the antiphospholipid antibody profile. J. Thromb. Haemost. 2013;11:1527–1531. doi: 10.1111/jth.12264. PubMed DOI

Pengo V., Ruffatti A., Legnani C., Gresele P., Barcellona D., Erba N., Testa S., Marongiu F., Bison E., Denas G., et al. Clinical course of high-risk patients diagnosed with antiphospholipid syndrome. J. Thromb. Haemost. 2010;8:237–242. doi: 10.1111/j.1538-7836.2009.03674.x. PubMed DOI

Pengo V., Denas G. Diagnostics and treatment of thrombotic antiphospholipid syndrome (APS): A personal perspec-tive. Thromb. Res. 2018;169:35–40. doi: 10.1016/j.thromres.2018.07.011. PubMed DOI

Tripodi A. Laboratory Testing for Lupus Anticoagulants: A Review of Issues Affecting Results. Clin. Chem. 2007;53:1629–1635. doi: 10.1373/clinchem.2007.089524. PubMed DOI

Chaturvedi S., McCrae K.R. Diagnosis and management of the antiphospholipid syndrome. Blood Rev. 2017;31:406–417. doi: 10.1016/j.blre.2017.07.006. PubMed DOI PMC

Liu T., Gu J., Wan L., Hu Q., Teng J., Liu H., Cheng X., Ye J., Su Y., Sun Y., et al. “Non-criteria” antiphospholipid antibodies add value to antiphospholipid syndrome diagnoses in a large Chinese cohort. Arthritis Res. 2020;22:1–11. doi: 10.1186/s13075-020-2131-4. PubMed DOI PMC

Choi H., Ahn S.S., Song J.J., Park Y., Song J., Lee S.-W. Anti-phospholipid antibody syndrome occurrence in patients with persistent anti-phospholipid antibodies. Rheumatol. Int. 2019;39:1359–1367. doi: 10.1007/s00296-019-04318-4. PubMed DOI

De Groot P.G., Meijers J.C. β(2)-Glycoprotein I: Evolution, structure and function. J. Thromb. Haemost. 2011;9:1275–1284. doi: 10.1111/j.1538-7836.2011.04327.x. PubMed DOI

Misasi R., Capozzi A., Longo A., Recalchi S., Lococo E., Alessandri C., Conti F., Valesini G., Sorice M. “New” antigenic targets and methodological approaches for refining laboratory diagnosis of antiphospho-lipid syndrome. J. Immunol. Res. 2015;2015:858542. doi: 10.1155/2015/858542. PubMed DOI PMC

Chayoua W., Kelchtermans H., Moore G.W., Musiał J., Wahl D., De Laat B., Devreese K.M.J. Identification of high thrombotic risk triple-positive antiphospholipid syndrome patients is dependent on anti-cardiolipin and anti-β2glycoprotein I antibody detection assays. J. Thromb. Haemost. 2018;16:2016–2023. doi: 10.1111/jth.14261. PubMed DOI

Decavele A.S., Schouwers S., Devreese K.M. Evaluation of three commercial ELISA kits for anticardiolipin and anti-β2-glycoprotein I antibodies in the laboratory diagnosis of the antiphospholipid syndrome. Int. J. Lab. Hematol. 2011;33:97–108. doi: 10.1111/j.1751-553X.2010.01259.x. PubMed DOI

Gebhart J., Posch F., Koder S., Quehenberger P., Perkmann T., Kuessel L., Ay C., Pabinger I. High risk of adverse pregnancy outcomes in women with a persistent lupus anticoagulant. Blood Adv. 2019;3:769–776. doi: 10.1182/bloodadvances.2018026948. PubMed DOI PMC

Pierangeli S.S., Favaloro E.J., Lakos G., Meroni P.L., Tincani A., Wong R.C., Harris E.N. Standards and reference materials for the anticardiolipin and anti-β2glycoprotein I assays: A report of recommendations from the APL Task Force at the 13th International Congress on Antiphospholipid Antibodies. Clin. Chim. Acta. 2012;413:358–360. doi: 10.1016/j.cca.2011.09.048. PubMed DOI

Willis R., Lakos G., Harris E.N. Standardization of Antiphospholipid Antibody Testing—Historical Perspectives and Ongoing Initiatives. Semin. Thromb. Hemost. 2014;40:172–177. doi: 10.1055/s-0033-1364207. PubMed DOI

Janek D., Slavik L., Ulehlova J., Krcova V., Hlusi A., Prochazkova J. Validation of a New Panel of Automated Chemiluminescence Assays for Anticardiolipin Antibodies in the Screening for Antiphospholipid Syndrome. Clin. Lab. 2016;62:1309–1315. doi: 10.7754/Clin.Lab.2015.151129. PubMed DOI

Meroni P.L., Tincani A., Harris E.N., Valesini G., Hughes G.R., Balestrieri G. The pathophysiology of anti-phospholipid antibodies. Clin. Exp. Rheumatol. 1989;7:81–84. PubMed

Yin D., Chayoua W., Kelchtermans H., de Groot P.G., Moore G.W., Gris J.C., Zuily S., Musial J., de Laat B., Devreese K.M.J. Detection of anti-domain I antibodies by chemiluminescence enables the identification of high-risk an-tiphospholipid syndrome patients: A multicenter multiplatform study. J. Thromb. Haemost. 2020;18:463–478. doi: 10.1111/jth.14682. PubMed DOI

Mahler M., Norman G.L., Meroni P.L., Khamashta M. Autoantibodies to domain 1 of β2 glycoprotein 1: A promising candidate biomarker for risk manage-ment in antiphospholipid syndrome. Autoimmun. Rev. 2012;12:313–317. doi: 10.1016/j.autrev.2012.05.006. PubMed DOI

Radin M., Cecchi I., Roccatello D., Meroni P.L., Sciascia S. Prevalence and Thrombotic Risk Assessment of Anti-β2 Glycoprotein I Domain I Antibodies: A Systematic Review. Semin. Thromb. Hemost. 2017;44:466–474. doi: 10.1055/s-0037-1603936. PubMed DOI

Tonello M., Mattia E., Del Ross T., Favaro M., Calligaro A., Hoxha A., Bison E., Pengo V., Ruffatti A. Clinical value of anti-domain I-β2Glycoprotein 1 antibodies in antiphospholipid antibody carriers. A single centre, prospective observational follow-up study. Clin. Chim. Acta. 2018;485:74–78. doi: 10.1016/j.cca.2018.06.037. PubMed DOI

Serrano M., Martinez-Flores J.A., Norman G.L., Naranjo L., Morales J.M., Serrano A. The IgA Isotype of Anti-β2 Glycoprotein I Antibodies Recognizes Epitopes in Domains 3, 4, and 5 That Are Located in a Lateral Zone of the Molecule (L-Shaped) Front. Immunol. 2019;10:1031. doi: 10.3389/fimmu.2019.01031. PubMed DOI PMC

Slavik L., Janek D., Ulehlova J., Krcova V., Hlusi A. Detection of Anti-Domain I β-2 Glycoprotein I Antibodies as New Potential Target in Antiphospholipid Syndrome Diagnosis. J. Hematol. Thrombo. Dis. 2017;5:276.

Pérez D., Tincani A., Serrano M., Shoenfeld Y., Serrano A. Antiphospholipid syndrome and IgA anti-β2-glycoprotein I antibodies: When Cinderella becomes a princess. Lupus. 2017;27:177–178. doi: 10.1177/0961203317738227. PubMed DOI

Morales J.M., Serrano M., Martinez-Flores J.A., Gainza F.J., Marcen R., Arias M., Escuin F., Pérez D., Andres A., Martínez M.A., et al. Pretransplant IgA-Anti-Beta 2 Glycoprotein I Antibodies as a Predictor of Early Graft Thrombosis after Renal Transplantation in the Clinical Practice: A Multicenter and Prospective Study. Front. Immunol. 2018;9:468. doi: 10.3389/fimmu.2018.00468. PubMed DOI PMC

Pericleous C., Ferreira I., Borghi O., Pregnolato F., McDonnell T., Garza-Garcia A., Driscoll P., Pierangeli S., Isenberg D., Ioannou Y., et al. Measuring IgA Anti-β2-Glycoprotein I and IgG/IgA Anti-Domain I Antibodies Adds Value to Current Serological Assays for the Antiphospholipid Syndrome. PLoS ONE. 2016;11:e0156407. doi: 10.1371/journal.pone.0156407. PubMed DOI PMC

Ruiz-García R., Serrano M., Martínez-Flores J.Á., Mora S., Morillas L., Martín-Mola M.Á., Morales J.M., Paz-Artal E., Serrano A. Isolated IgA Anti-β2 Glycoprotein I Antibodies in Patients with Clinical Criteria for Antiphospholipid Syndrome. J. Immunol. Res. 2014;2014:1–8. doi: 10.1155/2014/704395. PubMed DOI PMC

Vlagea A., Pascual-Salcedo D., Doforno R.Á., Lavilla P., Diez J., Merlano B.P., Cuesta M.V., Gil A. IgA anti-β2 glycoprotein I antibodies: Experience from a large center. Thromb. Res. 2018;162:38–43. doi: 10.1016/j.thromres.2017.12.007. PubMed DOI

Chayoua W., Yin D.-M., Kelchtermans H., Moore G.W., Gris J.-C., Musiał J., Zuily S., Cate H.T., De Laat B., Devreese K.M.J. Is There an Additional Value in Detecting Anticardiolipin and Anti-β2 glycoprotein I IgA Antibodies in the Antiphospholipid Syndrome? Thromb. Haemost. 2020;120:1557–1568. doi: 10.1055/s-0040-1714653. PubMed DOI

Devreese K.M.J. Testing for antiphospholipid antibodies: Advances and best practices. Int. J. Lab. Hematol. 2020;42:49–58. doi: 10.1111/ijlh.13195. PubMed DOI

Bizzaro N., Ghirardello A., Zampieri S., Iaccarino L., Tozzoli R., Ruffatti A., Villalta D., Tonutti E., Doria A. Anti-prothrombin antibodies predict thrombosis in patients with systemic lupus erythematosus: A 15-year longitudinal study. J. Thromb. Haemost. 2007;5:1158–1164. doi: 10.1111/j.1538-7836.2007.02532.x. PubMed DOI

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. Thromb. Haemost. 2014;111:354–364. doi: 10.1160/TH13-06-0509. PubMed DOI

Cifu’ A., Domenis R., Pistis C., Curcio F., Fabris M. Anti-β2-glycoprotein I and anti-phosphatidylserine/prothrombin antibodies exert similar pro-thrombotic effects in peripheral blood monocytes and endothelial cells. Autoimmun. Highlights. 2019;10:1–8. doi: 10.1186/s13317-019-0113-9. PubMed DOI PMC

Sciascia S., Radin M., Sanna G., Cecchi I., Roccatello D., Bertolaccini M.L. Clinical utility of the global anti-phospholipid syndrome score for risk stratification: A pooled analysis. Rheumatology. 2018;57:661–665. doi: 10.1093/rheumatology/kex466. PubMed DOI

Conti F., Capozzi A., Truglia S., Lococo E., Longo A., Misasi R., Alessandri C., Valesini G., Sorice M. The mosaic of “seronegative” antiphospholipid syndrome. J. Immunol. Res. 2014;2014:389601. doi: 10.1155/2014/389601. PubMed DOI PMC

Ho W.K., Rigano J. Prevalence of autoantibodies directed against prothrombin in unprovoked venous thromboembo-lism. J. Thromb. Thrombolysis. 2020;49:446–450. doi: 10.1007/s11239-020-02053-3. PubMed DOI

Shi H., Zheng H., Yin Y., Hu Q., Teng J., Sun Y., Liu H.-L., Cheng X., Ye J., Su Y., et al. Antiphosphatidylserine/prothrombin antibodies (aPS/PT) as potential diagnostic markers and risk predictors of venous thrombosis and obstetric complications in antiphospholipid syndrome. Clin. Chem. Lab. Med. 2017;56:614–624. doi: 10.1515/cclm-2017-0502. PubMed DOI

Rand J.H. Antiphospholipid Antibody-mediated Disruption of the Annexin-V Antithrombotic Shield: A Thrombogenic Mechanism for the Antiphospholipid Syndrome. J. Autoimmun. 2000;15:107–111. doi: 10.1006/jaut.2000.0410. PubMed DOI

Bertolaccini M.L., Amengual O., Atsumi T., Binder W.L., de Laat B., Forastiero R., Kutteh W.H., Lambert M., Matsubayashi H., Murthy V., et al. ‘Non-criteria’ aPL tests: Report of a task force and preconference workshop at the 13th International Congress on Antiphospholipid Antibodies, Galveston, TX, USA, April 2010. Lupus. 2011;20:191–205. doi: 10.1177/0961203310397082. PubMed DOI

Cañas F., Simonin L., Couturaud F., Renaudineau Y. Annexin A2 autoantibodies in thrombosis and autoimmune diseases. Thromb. Res. 2015;135:226–230. doi: 10.1016/j.thromres.2014.11.034. PubMed DOI

Ortona E., Capozzi A., Colasanti T., Conti F., Alessandri C., Longo A., Garofalo T., Margutti P., Misasi R., Khamashta M.A., et al. Vimentin/cardiolipin complex as a new antigenic target of the antiphospholipid syndrome. Blood. 2010;116:2960–2967. doi: 10.1182/blood-2010-04-279208. PubMed DOI

Arachchillage D.R.J., Efthymiou M., Mackie I.J., Lawrie A.S., Machin S.J., Cohen H. Anti-protein C antibodies are associated with resistance to endogenous protein C activation and a severe thrombotic phenotype in antiphospholipid syndrome. J. Thromb. Haemost. 2014;12:1801–1809. doi: 10.1111/jth.12722. PubMed DOI

Matyas G.R., Alving C.R. Antigen-specific enhancement of natural human IgG antibodies to phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol-4-phosphate, cholesterol, and lipid A by a liposomal vaccine containing lipid A. Vaccine. 2011;29:5137–5144. doi: 10.1016/j.vaccine.2011.05.042. PubMed DOI

Pignatelli P., Ettorre E., Menichelli D., Pani A., Violi F., Pastori D. Seronegative antiphospholipid syndrome: Refining the value of “non-criteria” antibodies for di-agnosis and clinical management. Haematologica. 2020;105:562–572. doi: 10.3324/haematol.2019.221945. PubMed DOI PMC

Korematsu S., Yamada H., Miyahara H., Ihara K. Increased levels of anti-phosphatidylcholine and anti-phosphatidylethanolamine antibodies in pediatric patients with cerebral infarction. Brain Dev. 2017;39:542–546. doi: 10.1016/j.braindev.2017.01.010. PubMed DOI

Alessandri C., Bombardieri M., Di Prospero L., Conigliaro P., Conti F., Labbadia G., Misasi R., Sorice M., Valesini G. Anti-lysobisphosphatidic acid antibodies in patients with antiphospholipid syndrome and systemic lupus erythematosus. Clin. Exp. Immunol. 2005;140:173–180. doi: 10.1111/j.1365-2249.2005.02727.x. PubMed DOI PMC

Castanon A., Pierre G., Willis R., Harris E.N., Papalardo E., Romay-Penabad Z., Schleh A., Jajoria P., Smikle M., DeCeulaer K., et al. Performance Evaluation and Clinical Associations of Immunoassays That Detect Antibodies to Nega-tively Charged Phospholipids Other Than Cardiolipin. Am. J. Clin. Pathol. 2018;149:401–411. doi: 10.1093/ajcp/aqy003. PubMed DOI PMC

Park H.S., Gu J.Y., Jung H.S., Kim H.K. Thrombotic Risk of Non-Criteria Anti-Phospholipid Antibodies Measured by Line Immunoassay: Superi-ority of Anti-Phosphatidylserine and Anti-Phosphatidic Acid Antibodies. Clin. Lab. 2019;65:171207. doi: 10.7754/Clin.Lab.2018.171207. PubMed DOI

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 Anticoagu-lant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Throm-bosis and Haemostasis. J. Thromb. Haemost. 2009;7:1737–1740. doi: 10.1111/j.1538-7836.2009.03555.x. PubMed DOI

McDonnell T.C.R., Willis R., Pericleous C., Ripoll V.M., Giles I.P., Isenberg D.A., Brasier A.R., Gonzalez E.B., Papalardo E., Romay-Penabad Z., et al. PEGylated Domain I of Beta-2-Glycoprotein I Inhibits the Binding, Coagulopathic, and Thrombo-genic Properties of IgG from Patients with the Antiphospholipid Syndrome. Front. Immunol. 2018;9:2413. doi: 10.3389/fimmu.2018.02413. PubMed DOI PMC

Linnemann B. Antiphospholipid syndrome—An update. Vasa. 2018;47:451–464. doi: 10.1024/0301-1526/a000723. PubMed DOI

Devreese K.M.J. How to Interpret Antiphospholipid Laboratory Tests. Curr. Rheumatol. Rep. 2020;22:1–11. doi: 10.1007/s11926-020-00916-5. PubMed DOI

Tripodi A., Chantarangkul V., Cini M., Devreese K., Dlott J.S., Giacomello R., Gray E., Legnani C., Martinuzzo M.E., Pradella P., et al. Variability of cut-off values for the detection of lupus anticoagulants: Results of an international multi-center multiplatform study. J. Thromb. Haemost. 2017;15:1180–1190. doi: 10.1111/jth.13678. PubMed DOI

Moore G.W. Current Controversies in Lupus Anticoagulant Detection. Antibodies. 2016;5:22. doi: 10.3390/antib5040022. PubMed DOI PMC

Cohen H., Mackie I.J., Devreese K.M.J. Clinical and laboratory practice for lupus anticoagulant testing: An Interna-tional Society of Thrombosis and Haemostasis Scientific and Standardization Committee survey. J. Thromb. Haemost. 2019;17:1715–1732. doi: 10.1111/jth.14560. PubMed DOI

Pradella P., Azzarini G., Santarossa L., Caberlotto L., Bardin C., Poz A., D’Aurizio F., Giacomello R. Cooperation experience in a multicentre study to define the upper limits in a normal population for the diagnostic assessment of the functional lupus anticoagulant assays. Clin. Chem. Lab. Med. 2013;51:379–385. doi: 10.1515/cclm-2012-0382. PubMed DOI

Aydin S. A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides. 2015;72:4–15. doi: 10.1016/j.peptides.2015.04.012. PubMed DOI

Kohl T.O., Ascoli C.A. Immunometric Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay. Cold Spring Harb. Protoc. 2017;2017:093724. doi: 10.1101/pdb.prot093724. PubMed DOI

Kohl T.O., Ascoli C.A. Indirect Immunometric ELISA. Cold Spring Harb. Protoc. 2017;2017:93708. doi: 10.1101/pdb.prot093708. PubMed DOI

Tebo A.E., Jaskowski T.D., Phansalkar A.R., Litwin C.M., Branch D.W., Hill H.R. Diagnostic Performance of Phospholipid-Specific Assays for the Evaluation of Antiphospholipid Syndrome. Am. J. Clin. Pathol. 2008;129:870–875. doi: 10.1309/6MPULFBL24FM9B50. PubMed DOI

Vanoverschelde L., Kelchtermans H., Musial J., de Laat B., Devreese K.M.J. Influence of anticardiolipin and anti-β2 glycoprotein I antibody cutoff values on antiphospholip-id syndrome classification. Res. Pract. Thromb. Haemost. 2019;3:515–527. doi: 10.1002/rth2.12207. PubMed DOI PMC

Bor M.V., Jacobsen I.S., Gram J.B., Sidelmann J.J. Revisiting the Phadia/EliA cut-off values for anticardiolipin and anti-β2-glycoprotein I antibodies: A sys-tematic evaluation according to the guidelines. Lupus. 2018;27:1446–1454. doi: 10.1177/0961203318776105. PubMed DOI

Zhou J., Hou X., Zhang H., Wang T., Cui L. The Clinical Performance of a New Chemiluminescent Immunoassay in Measuring Anti-β2 Glycoprotein 1 and Anti-Cardiolipin Antibodies. Med. Sci. Monit. 2018;24:6816–6822. doi: 10.12659/MSM.910369. PubMed DOI PMC

Yin D., De Laat B., Devreese K.M., Kelchtermans H. The clinical value of assays detecting antibodies against domain I of β2-glycoprotein I in the antiphospholipid syndrome. Autoimmun. Rev. 2018;17:1210–1218. doi: 10.1016/j.autrev.2018.06.011. PubMed DOI

Nakamura H., Oku K., Amengual O., Ohmura K., Fujieda Y., Kato M., Bohgaki T., Yasuda S., Atsumi T. First-Line, Non-Criterial Antiphospholipid Antibody Testing for the Diagnosis of Antiphospholipid Syndrome in Clinical Practice: A Combination of Anti-β(2)-Glycoprotein I Domain I and An-ti-Phosphatidylserine/Prothrombin Complex Antibodies Tests. Arthritis Care Res. 2018;70:627–634. doi: 10.1002/acr.23310. PubMed DOI

Chayoua W., Kelchtermans H., Moore G.W., Gris J.-C., Musial J., Wahl D., Zuily S., Gianniello F., Fontana P., Remijn J., et al. Detection of Anti-Cardiolipin and Anti-β2glycoprotein I Antibodies Differs between Platforms without Influence on Association with Clinical Symptoms. Thromb. Haemost. 2019;119:797–806. doi: 10.1055/s-0039-1679901. PubMed DOI

Salma N., Julie L., Boutahar B., Sylvie L.N., Eleonore B., Fabien L.N., Elisabeth P., Sandrine J.J., Francis C., Sophie H., et al. Thrombotic risk assessment and analytical performance of the chemiluminescent analyzer IDS-iSYS for the detection of anti-cardiolipin and anti-β2 glycoprotein I autoantibodies. Clin. Immunol. 2018;194:92–99. doi: 10.1016/j.clim.2018.07.006. PubMed DOI

Chayoua W., Kelchtermans H., Gris J.C., Moore G.W., Musiał J., Wahl D., de Groot P.G., de Laat B., Devreese K.M.J. The (non-)sense of detecting anti-cardiolipin and anti-β2glycoprotein I IgM antibodies in the antiphos-pholipid syndrome. J. Thromb. Haemost. 2020;18:169–179. doi: 10.1111/jth.14633. PubMed DOI

Grossi V., Infantino M., Benucci M., Gobbi F.L., Bandinelli F., Damiani A., Bodio C., Borghi M.O., Mahler M., Aure M.A., et al. Two Novel Technologies for the Detection of Anti-cardiolipin and Anti β2–Glycoprotein Antibodies in the Real Life: Chemiluminescent in Comparison to the Addressable Laser Bead Immunoassays. Immunol. Investig. 2019;49:58–68. doi: 10.1080/08820139.2019.1647233. PubMed DOI

Bevers E.M., Zwaal R.F., Willems G.M. The effect of phospholipids on the formation of immune complexes between autoantibodies and β2-glycoprotein I or prothrombin. Clin. Immunol. 2004;112:150–160. doi: 10.1016/j.clim.2004.02.009. PubMed DOI

Egerer K., Roggenbuck D., Buettner T., Lehmann B., Kohn A., Von Landenberg P., Hiemann R., Feist E., Burmester G.-R., Dorner T. Single-step autoantibody profiling in antiphospholipid syndrome using a multi-line dot assay. Arthritis Res. Ther. 2011;13:R118. doi: 10.1186/ar3421. PubMed DOI PMC

Thaler M.A., Bietenbeck A., Steigerwald U., Büttner T., Schierack P., Lindhoff-Last E., Roggenbuck D., Luppa P.B. Evaluation of the sensitivity and specificity of a novel line immunoassay for the detection of criteria and non-criteria antiphospholipid antibodies in comparison to established ELISAs. PLoS ONE. 2019;14:e0220033. doi: 10.1371/journal.pone.0220033. PubMed DOI PMC

Roggenbuck D., Borghi M.O., Somma V., Büttner T., Schierack P., Hanack K., Grossi C., Bodio C., Macor P., von Landenberg P., et al. Antiphospholipid antibodies detected by line immunoassay differentiate among patients with an-tiphospholipid syndrome, with infections and asymptomatic carriers. Arthritis Res. Ther. 2016;18:111. doi: 10.1186/s13075-016-1018-x. PubMed DOI PMC

Nalli C., Somma V., Andreoli L., Büttner T., Schierack P., Mahler M., Roggenbuck D., Tincani A. Anti-phospholipid IgG antibodies detected by line immunoassay differentiate patients with an-ti-phospholipid syndrome and other autoimmune diseases. Auto Immun. Highlights. 2018;9:6. doi: 10.1007/s13317-018-0106-0. PubMed DOI PMC

Conti F., Alessandri C., Sorice M., Capozzi A., Longo A., Garofalo T., Misasi R., Bompane D., Hughes G.R.V., Khamashta M.A., et al. Thin-layer chromatography immunostaining in detecting anti-phospholipid antibodies in seronegative anti-phospholipid syndrome. Clin. Exp. Immunol. 2011;167:429–437. doi: 10.1111/j.1365-2249.2011.04532.x. PubMed DOI PMC

Conti F., Alessandri C., Spinelli F., Capozzi A., Martinelli F., Recalchi S., Misasi R., Valesini G., Sorice M. TLC immunostaining for detection of “antiphospholipid” antibodies. Methods Mol. Biol. 2014;1134:95–101. PubMed

Sorice M., Griggi T., Circella A., Garofalo T., D’Agostino F., Pittoni V., Pontieri G., Lenti L., Valesini G. Detection of antiphospholipid antibodies by immunostaining on thin layer chromatography plates. J. Immunol. Methods. 1994;173:49–54. doi: 10.1016/0022-1759(94)90282-8. PubMed DOI

Albay A., Esen B.A., Pericleous C., Wincup C., Giles I., Rahman A., McDonnell T. Domain I of β2GPI is capable of blocking serum IgA antiphospholipid antibodies binding in vitro: An effect enhanced by PEGylation. Lupus. 2019;28:893–897. doi: 10.1177/0961203319851571. PubMed DOI PMC

Cervera R., Conti F., Doria A., Iaccarino L., Valesini G. Does seronegative antiphospholipid syndrome really exist? Autoimmun. Rev. 2012;11:581–584. doi: 10.1016/j.autrev.2011.10.017. PubMed DOI

Conti F., Andreoli L., Crisafulli F., Mancuso S., Truglia S., Tektonidou M.G. Does seronegative obstetric APS exist? “pro” and “cons”. Autoimmun. Rev. 2019;18:102407. doi: 10.1016/j.autrev.2019.102407. PubMed DOI

Truglia S., Capozzi A., Mancuso S., Recalchi S., Spinelli F.R., Perricone C., De Carolis C., Manganelli V., Riitano G., Garofalo T., et al. A Monocentric Cohort of Obstetric Seronegative Anti-Phospholipid Syndrome. Front. Immunol. 2018;9:1678. doi: 10.3389/fimmu.2018.01678. PubMed DOI PMC

Salle V. Seronegative antiphospholipid syndrome: Myth or reality? Rev. Med. Interne. 2020;41:265–274. doi: 10.1016/j.revmed.2020.02.005. PubMed DOI

Fabris M., Giacomello R., Poz A., Pantarotto L., Tanzi N., Curcio F., Tonutti E. The introduction of anti-phosphatidylserine/prothrombin autoantibodies in the laboratory diagnostic process of anti-phospholipid antibody syndrome: 6 months of observation. Autoimmun. Highlights. 2014;5:63–67. doi: 10.1007/s13317-014-0061-3. PubMed DOI PMC

Meroni P.L., Chighizola C.B., Rovelli F., Gerosa M. Antiphospholipid syndrome in 2014: More clinical manifestations, novel pathogenic players and emerging biomarkers. Arthritis Res. Ther. 2014;16:209. doi: 10.1186/ar4549. PubMed DOI PMC