Variability of anti-β₂ glycoprotein I antibodies measurement by commercial assays

Summary

The aim of this study was to evaluate the agreement in assay results between commercial kits for the measurement of anti- β2glycoprotein I antibodies. Ten manufacturers provided one IgG and one IgM kit to three testing centres. Samples from patients with primary (n=13) or secondary (n=3) antiphospholipid syndrome (APS), from lupus patients without APS features (n=6) and from normal individuals (n=2) were tested in the three centres according to manufacturers’ instructions. Dilutions in normal serum of a pool made from positive patients’ samples (Forum Calibrators) and dilutions of humanized monoclonal antibodies (MoAbs) were used as additional calibrators. The calibration curves obtained with each calibrator differed widely between kits. The rate of positivity of patients’ samples varied from 7 to 16 for IgG and from 2 to 17 for IgM, depending on the kit. Perfect agreement occurred in 12/22 samples for IgG and 5/22 samples for IgM. Samples from normals were found negative by all kits. Between kits, cutoff values varied up to five fold when expressed in Forum Calibrators arbitrary units and up to three fold when expressed in MoAbs equivalents. Examination of discrepant samples indicated that about half of the discrepancies, scoring 8:2 and 9:1, involved the same few kits. In highly discrepant samples, some kits appeared as high responders as compared to others. In conclusion, with the exception of a few kits, agreement in assay results was acceptable. In conclusion, additional efforts are however necessary, especially concerning the way to assess the cutoff point and the adoption of a reference calibrator, in order to improve standardization of the assays.

Keywords

Anti-β2glycoprotein I antibodies - assays - standardization - antiphospholipid antibodies

• References

• 1 Wilson WA, Gharavi AE, Koike T. et al. International consensus statement on preliminary classification criteria for definite Antiphospholipid syndrome.. Arthritis Rheum 1999; 42: 1309-11.
• 2 Galli M, Luciani D, Bertolini G. et al. Lupus anticoagulants are stronger risk factors for thrombosis than anticardiolipin antibodies in the antiphospholipid syndrome: a systematic review of the literature.. Blood 2003; 101: 1827-32.
• 3 Galli M, Barbui T. Antiphospholipid antibodies and thrombosis: strength of association.. Hematol J 2003; 4: 180-6.
• 4 Galli M, Barbui T. Antiphospholipid antibodies and pregnancy.. Best Pract & Res Clin Haematol 2003; 16: 211-25.
• 5 Brandt JT, Triplett DA, Alving B. et al. Criteria for the diagnosis of lupus anticoagulants: an update. On behalf of the Subcommittee on Lupus Anticoagulant/ Antiphospholipid Antibody of the Scientific and Standardisation Committee of the ISTH.. Thromb Haemost 1995; 74: 1185-90.
• 6 Jennings I, Greaves M, Mackie IJ. et al. Lupus anticoagulant testing: improvements in performance in a UK NEQAS proficiency testing exercise after dissemination of national guidelines on laboratory methods.. Br J Haematol 2002; 119: 364-9.
• 7 Arnout J, Meijer P, Vermylen J. 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 beta2-glycoprotein I.. Thromb Haemost 1999; 81: 929-34.
• 8 Reber G, Arvieux J, Comby E. et al. Multicenter evaluation of nine commercial kits for the quantitation of anticardiolipin antibodies.. Thromb Haemost 1995; 73: 444-52.
• 9 Favaloro EJ, Silvestrini R. Assessing the usefulness of anticardiolipin antibody assays: a cautious approach is suggested by high variation and limited consensus in multilaboratory testing.. Am J Clin Pathol 2002; 118: 548-57.
• 10 Harris EN. The second international anti-cardiolipin standardization workshop/The Kingston antiphospholipid antibody (KAPS) study group.. Am J Clin Pathol 1990; 94: 476-84
• 11 Tincani A, Allegri F, Sanmarco M. et al. Anticardiolipin antibody assay: a methodological analysis for a better consensus in routine determinations. A cooperative project of the European Antiphospholipid Forum.. Thromb Haemost 2001; 86: 575-83.
• 12 Viard JP, Amoura Z, Bach JF. Association of antibeta 2 glycoprotein I antibodies with lupus-type circulating anticoagulant and thrombosis in systemic lupus erythematosus.. Am J Med 1992; 93: 181-6.
• 13 Galli M, Luciani B, Bertolini G. et al. Anti-β2-glycoprotein I, antiprothrombin antibodies and the risk of thrombosis in the antiphospholipid syndrome.. Blood 2003; 102: 2717-23.
• 14 Pengo V. Communication. 48th Annual SSC meeting. Boston MA; USA: July 18 2002. See annual SSC reports at the ISTH website http://www.med.unc.edu/isth
• 15 Nash MJ, Camilleri RS, Kunka S. et al. The anticardiolipin assay is required for sensitive screening for antiphospholipid antibodies.. J Thromb Haemost 2004; 2: 1077-81.
• 16 Reber G, Schousboe I, Tincani A. et al. Inter-laboratory variability of anti-ß2-glycoprotein I measurement. A collaborative study in the frame of the European forum on antiphospholipid antibodies standardization group.. Thromb Haemost 2002; 88: 66-73.
• 17 Ichikawa K, Tsutsumi A, Atsumi T. et al. A chimeric antibody with the human γ1 constant region as a putative standard for assays to detect IgG ß2-glycoprotein I-dependent anticardiolipin and anti-ß2-glycoprotein I antibodies.. Arthritis Rheum 1999; 42: 2461-70.
• 18 Tsutsumi A, Ichikawa K, Matsuura E. et al. Heterogeneous behavior of anti-ß2-glycoprotein I antibodies on various commercially available enzyme immunoassay plates coated with ß2-glycoprotein I.. J Rheumatol 2000; 27: 391-6.
• 19 Iverson GM, Matsuura E, Victoria EJ. et al. The orientation of beta2GPI on the plate is important for the binding of anti-beta2GPI autoantibodies by ELISA.. J Autoimmunity 2002; 18: 289-97.
• 20 Roubey RA, Eisenberg RA, Harper MF. et al. Anticardiolipin autoantibodies recognize ß2-glycoprotein I in the absence of phospholipids. Importance of antigen density and bivalent binding.. J Immunol 1995; 154: 954-60.
• 21 Reddel SW, Wang YX, Krilis SA. Anti-beta2-glycoprotein I autoantibodies require an antigen density threshold, consistent with divalent binding.. Lupus 2003; 12: 37-45.
• 22 Brighton TA, Dai YP, Hogg PJ. et al. Microheterogeneity of beta-2 glycoprotein I: implications for binding to anionic phospholipids.. Biochem J 1999; 340: 59-67.
• 23 Schousboe I. Characterization of subfractions of beta 2-glycoprotein I: evidence for sialic acid microheterogeneity.. Int J Biochem 1983; 15: 35-44.
• 24 Gries A, Nimpf J, Wurm H. et al. Characterization of isoelectric subspecies of asialo-beta 2-glycoprotein I.. Biochem J 1989; 260: 531-4.
• 25 Audrain MA, Colonna F, Morio F. et al. Comparison of different kits in the detection of autoantibodies to cardiolipin and beta2glycoprotein 1.. Rheumatology 2004; 43: 181-5.
• 26 Reber G, Tincani A, Sanmarco M. et al. Proposals for the measurement of anti-beta2-glycoprotein I antibodies. Standardization group of the European Forum on Antiphospholipid Antibodies.. J Thromb Haemost 2004; 2: 1860-2.