Lupus Anticoagulant Testing in Europe: An Analysis of Results from the First European Concerted Action on Thrombophilia (ECAT) Survey Using Plasmas Spiked with Monoclonal Antibodies against Human β2-Glycoprotein I

J. Arnout; P. Meijer; J. Vermylen

doi:10.1055/s-0037-1614601

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1999; 81(06): 929-934
DOI: 10.1055/s-0037-1614601

Letters to the Editor

Schattauer GmbH

Lupus Anticoagulant Testing in Europe: An Analysis of Results from the First European Concerted Action on Thrombophilia (ECAT) Survey Using Plasmas Spiked with Monoclonal Antibodies against Human β₂-Glycoprotein I

J. Arnout

¹From the Center for Molecular and Vascular Biology, University of Leuven, Belgium and ECAT Foundation, International Thrombophilia External Quality Assessment Scheme, Leiden, The Netherlands

,

P. Meijer

¹From the Center for Molecular and Vascular Biology, University of Leuven, Belgium and ECAT Foundation, International Thrombophilia External Quality Assessment Scheme, Leiden, The Netherlands

,

J. Vermylen

¹From the Center for Molecular and Vascular Biology, University of Leuven, Belgium and ECAT Foundation, International Thrombophilia External Quality Assessment Scheme, Leiden, The Netherlands

› Author Affiliations

Abstract

Summary

Lupus anticoagulants (LA) are immunoglobulins directed to either prothrombin or Beta-2-glycoprotein I (β₂GPI) bound to phospholipids. Most patients with LA have both β₂GPI- and prothrombin-dependent antibodies. Several recent reports have shown that LA is more strongly associated with thrombosis than anticardiolipin antibodies (aCL). Therefore, an accurate detection of LA is of utmost importance in patients suspected of an antiphospholipid syndrome. We recently raised a series of murine monoclonal antibodies against human Beta-2-glycoprotein I (β₂GPI) with LA activity similar to affinity purified human β₂GPI-dependent LAs. A normal plasma pool, and the same pool spiked with LA positive anti-β₂GPI antibodies at two potency levels, were used as materials in an external quality assessment scheme organised by the European Concerted Action on Thrombosis (ECAT). Fifty nine laboratories participating in this trial were asked to test for the presence of a LA in the 3 samples submitted. The majority (82%) of the participants found the high potency LA sample to be positive. Only 37% of the laboratories considered the weak potency LA sample to be positive. The submission of a normal sample, a weakly positive sample and a clearly positive sample enabled us to compare the relative LA responsiveness of the different screening assays used. Clotting time ratios varied from 0.81 to 3.28 for sample B and from 0.66 to 5.32 for sample D. In general, the highest clotting time ratios were found with the dilute prothrombin time (dPT), the dilute Russell Viper Venom time (dRVVT) and the Kaolin Clotting time. The most frequently used screening tests were the aPTT and the dRVVT. With the various assay systems, LA responsiveness varied largely according to the reagents used. For the β₂GPI-dependent LA used in this study, PTT LA clearly showed the highest responsiveness among the aPTT reagents and Innovin among the dPT reagents. The present study also shows that many laboratories still rely on poorly responsive screening assays for their LA tests. Other laboratories rely on sensitive and more specific integrated test systems based on a sensitive screening assay with a low phospholipid content and a confirmatory test employing high phospholipid concentrations. The most used integrated system was dRVVT based. However, also here the LA responsiveness was largely reagent dependent.

In conclusion, many laboratories still rely on poorly responsive screening assays by which weakly positive LA samples are misdiagnosed. LA positive anti-β₂GPI moabs have a potential for the unlimited production of LA control specimens, that may help hemostasis laboratories choose more LA responsive assay systems and to assess intra-laboratory precision of their LA testing.

Full Text

References

References
1 Love PE, Santoro SA. Antiphospholipid antibodies: Anticardiolipin and the lupus anticoagulant in systemic lupus erythematosus (SLE) and in non-SLE disorders. Ann Intern Med 1990; 112: 682-98.
2 McNeil HP, Simpson RJ, Chesterman CN, Krilis SA. Antiphospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: beta-2-glycoprotein I (apolipoprotein H). Proc Natl Acad Sci USA 1990; 87: 4120-4.
3 Galli M, Comfurius P, Maassen C, Hemker HC, de Baets MH, van Breda-Vriesman PJC, Barbui T, Zwaal RFA, Bevers EM. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 1990; 335: 1544-7.
4 Matsuura E., Igarashi Y, Fujimoto M, Ichikawa K, Koike T. Anticardiolipin cofactor(s) and differential diagnosis of autoimmune disease. Lancet 1990; 336: 177-8.
5 Bevers EM, Galli M, Barbui T, Comfurius P, Zwaal RF. Lupus anticoagulant IgG"s (LA) are not directed to phospholipids only, but to a complex of lipid-bound human prothrombin. Thromb Haemost 1991; 66: 629-32.
6 Oosting JD, Derksen RH, Entjes HT, Bouma BN, de Groot PhG. Lupus anticoagulant activity is frequently dependent on the presence of beta 2-glycoprotein I. Thromb Haemost 1992; 67: 499-502.
7 Roubey RA, Pratt CW, Buyon JP, Winfield JB. Lupus anticoagulant activity of autoimmune antiphospholipid antibodies is dependent upon beta 2-glycoprotein I. J Clin Invest 1992; 90: 1100-4.
8 Galli M, Comfurius P, Barbui T, Zwaal RF, Bevers EM. Anticoagulant activity of beta 2-glycoprotein I is potentiated by a distinct subgroup of anticardiolipin antibodies. Thromb Haemost 1992; 68: 297-300.
9 Horbach DA, van Oort E, Derksen RHWM, de Groot PhG. The contribution of antiprothrombin-antibodies to lupus anticoagulant activity. Discrimination between functional and nonfunctional antiprothrombin antibodies. Thromb Haemost 1998; 79: 790-5.
10 Horbach DA, van Oort E, Donders RCJM, Derksen RHWM, de Groot PG. Lupus anticoagulant is the strongest risk factor for both venous and arterial thrombosis in patients with systemic lupus erythematosus. Comparison between different assays for the detection of antiphospholipid antibodies. Thromb Haemost 1996; 76: 916-24.
11 Swadzba J, Declerck LS, Stevens WJ. Association of anticardiolipin anti-β₂-Glycoprotein I, antiprothrombin antibodies, and lupusanticoagulants in patients with systemic lupus erythematosus with a history of thrombosis. J. Rheumatol 1997; 24: 1710-5.
12 Galli M, Ruggeri L, Barbui T. Differential effects of anti-β₂-Glycoprotein I and antiprothrombin antibodies on the anticoagulant activity of activated protein C. Blood 1998; 91: 1999-2004.
13 Exner T, Triplett DA, Taberner D, Machin SJ. Guidelines for testing and revised criteria for lupus anticoagulants. Thromb Haemost 1991; 65: 320-2.
14 Brandt JT, Triplett DA, Alving B, Scharrer I. Criteria for the diagnosis of lupus anticoagulants: an update. Thromb Haemost 1995; 74: 1185-90.
15 Roussi J, Roisin JP, Goguel A. Lupus anticoagulants. First French Inter-laboratory Etalanorme Survey. Am J Clin Pathol 1996; 105: 788-93.
16 Jennings I, Kitchen S, Woods TAL, Preston FE, Greaves M.. Clinically important inaccuracies in testing for the lupus anticoagulant: an analysis of results from three surveys of the UK national external quality assessment scheme (NEQAS) for blood coagulation. Thromb Haemost 1997; 77:: 934-7.
17 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: 1597-603.
18 Capel P, Arnout J, Cauchie P, Chatelin B, Criel A, David JL, Fondu P, Jochmans K, Lavenne E, Lust A, Van der Planken M. Comparative study of antiphospholipid antibody detection in eleven Belgian laboratories. Acta Clin Belg 1997; 52: 84-91.
19 Arnout J, Wittevrongel C, Vanrusselt M, Hoylaerts M, Vermylen J. Beta-2-glycoprotein I dependent lupus anticoagulants form stable bivalent antibody-beta-2-glycoprotein I complexes on phospholipid surfaces. Thromb Haemost 1998; 79: 79-86.
20 Willems GM, Janssen MP, Pelsers MMAL, Confurius P, Galli M, Zwaal RFA, Bevers EM. Role of divalency in the high-affinity binding of anti-cardiolipin antibody-β₂-glycoprotein I complexes to lipid membranes. Biochemistry 1996; 35: 13833-42.
21 Takeya H, Mori T, Gabazza EC, Kuroda K, Deguchi H, Matsuura E, Ichikawa K, Koike T, Suzuki K. Anti-β₂-Glycoprotein I (β₂GP I) monoclonal antibodies with lupus anticoagulant-like activity enhance the β₂GP I binding to phospholipids. J Clin Invest 1997; 99: 2260-8.
22 Arnout J, Vermylen J.. Lupus anticoagulant: influence on the international normalized ratio. Thromb Haemost 1999. (in press)
23 Arnout J., Vanrusselt M., Wittevrongel C., Vermylen J. Monoclonal antibodies against Beta-2-glycoprotein I: Use as reference material for lupus anticoagulant tests. Thromb Haemost 1998; 79: 955-8.
24 Lesperance B, David M, Rauch J, Infante-Rivard C, Rivard GE. Relative sensitivity of different tests in the detection of low titer lupus anticoagulants. Thromb Haemost 1988; 60: 217-9.
25 Lo SCL, Oldmeadow MJ, Howard MA, Firkin BG. Comparison of laboratory tests used for identification of the lupus anticoagulant. American Journal of Hematology 1989; 30: 213-20.
26 Martin BA, Branch DW, Rodgers GM. Sensitivity of the activated partial thromboplastin time, the dilute Russell’s viper venom time, and the kaolin clotting time for the detection of the lupus anticoagulant: a direct comparison using plasma dilutions. Blood Coagul Fibrinolysis 1996; 7: 31-8.
27 Mannucci PM, Canciani MT, Mari D, Meucci P.. The varied sensitivity of partial thromboplastin time reagents in the demonstration of the lupus-like anticoagulant. Scand J Haematol 1979; 22: 423-32.
28 Brandt JT, Triplett DA, Musgrave K, Orr C. The sensitivity of different coagulation reagents to the presence of lupus anticoagulant. Arch Pathol Lab Med 1987; 111: 120-4.
29 Adcock DM, Marlar RA. Activated partial thromboplastin time reagent sensitivity to the presence of the lupus anticoagulant. Arch Pathol Lab Med 1992; 116: 837-40.
30 Denis-Magdelaine A, Flahault A, Verdy E. Sensitivity of sixteen aPTT reagents for the presence of lupus anticoagulants. Haemostasis 1995; 25: 98-105.
31 Forastiero RR, Cerrato GS, Carreras LO. Evaluation of recently described tests for detection of the lupus anticoagulant. Thromb Haemost 1994; 72: 728-33.
32 Arnout J., Vanrusselt M., Huybrechts E., Vermylen J. Optimisation of the dilute prothrombin time for the detection of the lupus anticoagulant by use of a recombinant tissue thromboplastin. Br J Haematol 1994; 87: 94-9.