Assessment of antiphospholipid antibodies using enzyme-linked immunosorbent assay and chemiluminescent method

Antiphospholipid antibodies (aPL) are a sign of acquired thrombophilia and are associated with recurrent thrombosis and obstetric pathology. According 2006 classification criteria, serological markers of antiphospholipid syndrome (APS) include lupus anticoagulant, moderate and high levels of antibodies to cardiolipin (aСL) and â2-glycoprotein 1 (anti- â2-GP1) IgG and IgM. The task of standardizing aPL values remains unresolved, leading to variability in results.

Objective: to evaluate the comparability of IgG/IgM aCL and IgG/IgM anti- â2-GP1 measurement results using enzyme-linked immunosorbent assay (ELISA) and chemiluminescence (CLU) tests.

Material and methods. Peripheral blood from 192 patients (147 women and 45 men) was analyzed, 55 patients (29%) had primary APS, 12 (6%) had probable APS, 61 (32%) had systemic lupus erythematosus (SLE) and APS, and 64 (33%) had SLE without APS. IgG/IgM aCL and IgG/IgM anti- â2-GP1 were determined in all participants by ELISA. By CLU method IgG/IgM aCL were analyzed in 192 patients and IgG/IgM anti- â2-GP1 in 191 patients.

Results and discussion. The evaluation of the comparability of the results of ELISA and CLU revealed considerable discrepancies in the positive tests. Thus, in 16% of cases, a discrepancy was found in the levels of IgG aCL and IgM aCL by both methods (n=30 and n=31, respectively), in 18% in the level of IgG anti- â2-GP1 (n=34) and in 15% in the level of IgM anti- â2-GP1 (n=28).These discrepancies were to a greater extent associated with a more frequent detection of aPL in CLU when their values were negative in ELISA , indicating the greater information capacity of the CLU method.

However, a small number of patients were positive for aPL in ELISA but negative in CLU: 5 with IgG aCL, 4 with IgM aCL, 6 with IgG anti-â2-GP1, and 2 with IgM anti- â2-GP1 .

Conclusion. CLU is shown to be a more informative method for determining IgG aCL and IgG anti- â2-GP1 than ELISA (p<0.05).

1. Arachchillage DRJ, Laffan M. Pathogenesis and management of antiphospholipid syndrome. Br J Haematol. 2017 Jul;178(2): 181-195. doi: 10.1111/bjh.14632.

2. Aringer M, Costenbader K, Daikh D, et al. 2019 European League Against Rheumatism/American College of Rheumatology Classification Criteria for Systemic Lupus Erythematosus. Arthritis Rheumatol. 2019 Sep; 71(9):1400-1412. doi: 10.1002/art.40930.

3. Asherson RA. New subsets of the antiphospholipid syndrome in 2006: "PRE-APS" (probable APS) and microangiopathic antiphospholipid syndromes ("MAPS"). Autoimmun Rev. 2006 Dec;6(2):76-80. doi: 10.1016/j.autrev.2006.06.008.

4. Atsumi T, Amengual O, Yasuda S, Koike T. Antiprothrombin antibodies – are they worth assaying? Thromb Res. 2004;114(5-6):533-8. doi: 10.1016/j.thromres.2004.08.024.

5. Miyakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost. 2006 Feb;4(2):295-306. doi: 10.1111/j.1538-7836.2006.01753.x.

6. Bertolaccini ML, Atsumi T, Koike T, et al. Antiprothrombin antibodies detected in two different assay systems. Prevalence and clinical significance in systemic lupus erythematosus. Thromb Haemost. 2005 Feb;93(2):289-97. doi: 10.1160/TH04-06-0382.

7. Bidot CJ, Jy W, Horstman LL, et al. Factor VII/VIIa: a new antigen in the anti-phospholipid antibody syndrome. Br J Haematol. 2003 Feb;120(4):618-26. doi: 10.1046/j.1365-2141.2003.04161.x.

8. 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 Jul;8(7):1540-6. doi: 10.1111/j.1538-7836.2010.03857.x. Epub 2010 Mar 16.

9. Alexandrova EN, Novikov AA, Reshetnyak TM, et al. Antibodies to 2-glycoprotein 1 and antibodies to cardiolipin in antiphospholipid syndrome: sensitivity and specificity analysis. Klinicheskaya meditsina. 2003;(9):25-31. (In Russ.).

10. Rebrova OYu. Statistical analysis of medical data. Application of STATISTICA package of applied programs. Moscow: MediaSfera; 2002.

11. https://telemedai.ru/biblioteka-dokumentov/klinicheskie-ispytaniya-programmnogoobespecheniya-na-osnove-intellektualnyhtehnologij-luchevaya-diagnostika

12. Barbhaiya M, Zuily S, Naden R, et al. ACR/EULAR APS Classification Criteria Collaborators. The 2023 ACR/EULAR Antiphospholipid Syndrome Classification Criteria. Arthritis Rheumatol. 2023 Oct;75(10): 1687-1702. doi: 10.1002/art.42624.

13. Devreese KMJ, Bertolaccini ML, Branch DW, et al. An update on laboratory detection and interpretation of antiphospholipid antibodies for diagnosis of antiphospholipid syndrome: guidance from the ISTH-SSC Subcommittee on Lupus Anticoagulant/Anti phospholipid Antibodies. J Thromb Haemost. 2024 Nov 5:S1538-7836(24)00638-X. doi: 10.1016/j.jtha.2024.10.022.

14. Atsumi T, Chighizola CB, Fujieda Y, et al. 16th International congress on antiphospholipid antibodies task force report on antiphospholipid syndrome laboratory diagnostics and trends. Lupus. 2023 Dec;32(14):1625-1636. doi: 10.1177/09612033231211820.

15. Forastiero R, Papalardo E, Watkins M, et al. Evaluation of different immunoassays for the detection of antiphospholipid antibodies: report of a wet workshop during the 13th International Congress on Antiphospholipid Antibodies. Clin Chim Acta. 2014 Jan 20;428: 99-105. doi: 10.1016/j.cca.2013.11.009.

16. Reber G, Boehlen F, de Moerloose P. Technical aspects in laboratory testing for antiphospholipid antibodies: is standardization an impossible dream? Semin Thromb Hemost. 2008 Jun;34(4):340-6. doi: 10.1055/s-0028-1085476.

17. Sciascia S, Montaruli B, Infantino M. Antiphospholipid antibody testing. Med Clin (Barc). 2024 Aug;163 Suppl 1:S4-S9. doi: 10.1016/j.medcli.2024.06.002.

18. Van Hoecke F, Persijn L, Decavele AS, 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 Dec;34(6):630-40. doi: 10.1111/j.1751-553X.2012.01448.x.

19. Devreese KMJ, de Groot PG, de Laat B, et al. Guidance from the Scientific and Standardization Committee for lupus anticoagulant/antiphospholipid antibodies of the International Society on Thrombosis and Haemostasis: Update of the guidelines for lupus anticoagulant detection and interpretation. J Thromb Haemost. 2020 Nov;18(11): 2828-2839. doi: 10.1111/jth.15047.

20. Tkachenko OYu., Lapin SV, Lazareva NM, et al. Comparative analysis of the informativeness of test systems from different manufacturers to determine antiphospholipid antibodies for diagnosis of antiphospholipid syndrome. Klinicheskaya laboratornaya diagnostika. 2017;62(1):40-4. (in Russ.).

21. Zhang S, Wu Z, Li P, et al. Evaluation of the Clinical Performance of a Novel Chemiluminescent Immunoassay for Detection of Anticardiolipin and Anti-Beta2-Glycoprotein 1 Antibodies in the Diagnosis of Antiphospholipid Syndrome. Medicine (Baltimore). 2015 Nov;94(46):e2059. doi: 10.1097/MD.0000000000002059.

22. Borghi MO, Beltagy A, Garrafa E, et al. Anti-Phospholipid Antibodies in COVID-19 Are Different From Those Detectable in the Anti-Phospholipid Syndrome. Front Immunol. 2020 Oct 15;11:584241. doi: 10.3389/fimmu.2020.584241.

23. Oku K, Amengual O, Kato M, et al. Significance of fully automated tests for the diagnosis of antiphospholipid syndrome. Thromb Res. 2016 Oct;146:1-6. doi: 10.1016/j.thromres.2016.08.018.

24. Capozzi A, Lococo E, Grasso M, et al. Detection of antiphospholipid antibodies by automated chemiluminescence assay. J Immunol Methods. 2012 May 31;379(1-2):48-52. doi: 10.1016/j.jim.2012.02.020.

25. Volkova MV, Kunder EV, Generalov A., Roggenbuk D. Antiphos-pholipid antibodies: modern ideas about pathogenetic action and laboratory diagnosis. Vestnik Vitebskogo gosudarstvennogo meditsinskogo universiteta 2015;(14-3):6-15. (In Russ.).