As published in CLI November 2005 A utoimmunity Autoantibodies in the diagnosis of APS by Dr Rainer Woehrle and Prof. Dr Klaus Helmke Some patients with clinical symptoms suggestive of antiphospholipid syndrome (APS) nevertheless test negative with the well standardised assays that detect antibodies against cardiolipin/ß2-glycoprotein-I complex. In such cases we recommend testing for antibodies against the human prothrombinphosphatidylserine complex, phosphatidylethanolamine, and against phosphatidylinositol with human ß2-GPI present as a co-factor in the assay. In patients who are still negative for these phospholipid antibodies, testing for antibodies against phosphatidylcholine, sphingomyelin, thrombin and annexin V may be helpful to identify individuals who would benefit from anticoagulant therapy. Tests should always detect IgG and IgM isotypes. The clinical manifestations of antiphospholipid syndrome (APS), which are mainly arterial and venous thrombosis and obstetric complications, are generally closely correlated with laboratory results. Historically the first indications of the existence of APS were false positive tests for syphilis: patients with Systemic Lupus Erythematosus (SLE) as well as some healthy subjects had positive venereal disease research laboratory (VDRL) tests in the absence of syphilis. Furthermore, many of these individuals showed prolonged coagulation times in in vitro assays. In 1952 a coagulation inhibitor was identified, the so called lupus anticoagulant (LAC), which in contrast enhances coagulation in vivo. Cardiolipin (CL), the negatively charged phospholipid present in the mitochondrial membrane, is a major component of the VDRL test. For this reason, an assay was developed in the early 1980s for the detection of anti-Cardiolipin (aCL) antibodies in plasma. Further investigations in 1990 showed that aCL-antibodies require a co-factor to bind to the cardiolipin targets; this co-factor is the plasma protein ß2-glycoprotein-I (ß2-GPI). In the meantime ELISAs for the detection of antibodies against the CL/ß2-GPI complex have become well standardised and accepted. These tests form the backbone of the laboratory diagnosis of APS [Table 1]. In addition to CL, antibodies against other phospholipids and proteins have been identified in the plasma of patients with clinical manifestations of APS [Table 2]. The aim of this review is to evaluate the diagnostic role of these antibodies against various phospholipid/protein complexes. Antibodies against cardiolipin and ß2-glycoprotein-I These antibodies bind to a cryptic epitope on the fifth domain of ß2-glycoprotein-I. When this serum protein binds to a surface containing cardiolipin (e.g. biological cell membranes or coated ELISA plates), the hidden epitope is exposed, allowing any aCLantibodies to bind [1]. Test systems for the detection of aCL-antibodies should therefore always contain sufficient amounts of ß2-GPI as a co-factor. These well standardised ELISAs have a reasonably high sensitivity (80-90%) for the diagnosis of APS patients. IgG antibodies are found most frequently, but a number of APS patients are only positive for the IgM isotypes. Testing for both IgG and IgM isotypes is therefore necessary. To facilitate the comparability of results between different test systems and laboratories, the test results should be reported by range of positivity in addition to the standardised G phospholipid (GPL) and M phospholipid (MPL) units [2]. Because the antibodies bind to the CL/ß2-GPI complex, tests detecting antibodies against ß2-GPI alone have a lower sensitivity (40-90%), especially if they use bovine ß2-GPI. On the other hand, conventional aCL antibody assays often also detect "ß2GPI-independent aCL" that are not associated with thromboembolic complications. Utilising anti-ß2-GPI antibody tests increases the specificity for patients with APS from 60% to 80%. As ß2-GPI antibodies of some patients are species-specific, the assay should use human ß2-GPI [3]. Antibodies against phosphatidylserine and prothrombin As with antibodies against CL and ß2-GPI, the binding of antibodies to prothrombin is improved if the prothrombin is itself bound with anionic phospholipids, especially phosphatidylserine (PS). While the levels of these antibodies showed a strong association with pregnancy loss [4], a significant correlation with thrombotic occlusions of blood vessels could nevertheless not be demonstrated in all studies. Testing for these antibodies should therefore be particularly carried out in women with pregnancy losses of unknown origin who are negative for aCL-antibodies and LAC. Since a high percentage of these antibodies have species-specificity for human protein, the assays should contain human prothrombin [5]. Antibodies against phosphatidylethanloamine In contrast to CL and the other anionic phospholipids, phosphatidylethanolamine (PE) is a zwitterionic phospholipid which is normally present in the outer layer of cell membranes. Antibodies against this phospholipid do not bind to phosphatidylethanolamine itself, but rather to a complex of PE and certain proteins in bovine serum, e.g. kininogens and kininogen-binding proteins; ß2-GPI is not involved. Table 1. Sapporo criteria for the diagnosis of APS, revised during the 11th International Congress on Antiphospholipid Antibodies in Sydney in 2004. The definite diagnosis of APS requires the presence of at least one clinical manifestation and one positive laboratory test result. Table 2. Phospholipid/Protein complexes as targets of antiphospholipid antibodies. To date studies on larger patient cohorts have not demonstrated the clinical utility of testing for PE antibodies, but many anecdotal case reports and antibody studies support the suggestion that there is a correlation between anti-PE antibodies and thromboembolic events [6]. Our own investigations also show this. It is reasonable therefore to consider anti-PE-testing in patients suspected of having APS, but who are negative with the standard aCL-ELISA. Antibodies against phophatidylinositol Since the late 1990s the significant association between the presence of antibodies to another anionic phospholipid, namely phosphatidyl-inositol (PI), and cerebral ischaemia especially in young patients, has been well documented [7]. In a cohort of 162 patients with autoimmune disease we also found anti-PI-antibodies to be significantly elevated in individuals with a history of cerebral ischaemia. Multivariate analyses showed that these antibodies were the only ones that were independently associated with stroke. Other groups have also demonstrated the association between anti-PI antibodies and clinical manifestations of APS. As with anti-PE antibodies, testing for anti-PI antibodies should be considered when patients are suspected of APS, as well as when there are negative test results with the standard aCL-ELISA, especially in young patients with cerebral ischaemia of undetermined cause. Since the binding of anti-PI antibodies is improved by the presence of ß2-GPI as a co-factor in the assay, test systems should contain human ß2-GPI. Antibodies against phosphatidylcholine or sphingomyelin In a study of 156 patients with autoimmune disease who were tested for antibodies against different phospholipids, 61 individuals were found to be negative for IgG antibodies against CL, ß2-GPI A utoimmunity or PE. In the latter group, IgG-antibodies against the anionic phospholipids phosphatidyl-choline (PC) and sphingomyelin (SM) were significantly elevated in patients with typical clinical symptoms of APS [8] using test systems that did not contain ß2-GPI as a co-factor. We therefore recommend testing for antibodies against PC and SM in individuals suspected of having APS who are negative for the tests discussed above. The test systems should not contain ß2-GPI as a co-factor. Data in the literature on the clinical utility of testing for anti-PC antibodies are contradictory. So far, anti-SM-antibodies have rarely been tested. Antibodies against thrombin and annexin V During recent years, antibodies specific for the coagulation factor thrombin have come under increased scrutiny. These antibodies are present in a number of APS patients and are associated with thromboembolic complications, mainly arterial thrombosis [9]. We also found 7 out of 8 women with unexplained pregnancy losses to be positive for antibodies against thrombin [10]. These results are preliminary and have to be verified in larger patient cohorts. Nevertheless in research projects, testing for thrombin antibodies should be considered in patients with suspected APS and whose results with the tests mentioned above are negative. The plasma protein annexin V (A5) binds to PS-rich surfaces such as the membrane of endothelial cells and placental cells forming an anticoagulant layer. Theoretically, it could be expected that disruption of this "anticoagulant shield" by antibodies could lead to an elevated risk of thromboembolic events. Clinical studies have verified that this theory is correct, with a significantly higher risk of thrombotic events being present in patients with systemic autoimmune disease. As published in CLI November 2005 Antiphospholipid Antibody study (KAPS) group. Am J Clin Pathol 1994; 94: 476-484. 3. Roubey RAS. Species-specific Autoantibodies to Human ß2GPI. In: Khamashta MA (Editor). Hughes Syndrome: Antiphospholipid Syndrome. Springer, 2000; 256-257. 4. Matthias T, von Landenberg P, Zaech J, Blank M, Shoenfeld Y. Anti- prothrombin antibodies are highly associated with pregnancy loss in patients with antiphospholipid syndrome. Table 3. Indications for anti-phospholipid-antibody testing. Annals of Hematology 2003; 82: S60. 5. Bertolaccini ML, Amengual O, Atsumi T. Antiprothrombin Antibodies. In: Khamashta MA (Editor). Hughes Syndrome: Antiphospholipid Syndrome. Springer, 2000; 245-252. 6. Roubey RAS. Antibodies Detected in Immunoassays Using Phosphatidylethanolamine. In: Khamashta MA (Editor). Hughes Syndrome: Antiphospholipid Syndrome. Springer, 2000; 256. 7. Toschi V, Motta A, Castelli C, Paracchini ML, Zerbi, Gibelli A. High prevalence of antiphosphatidylinositol antibodies in young patients with cerebral ischemia of undetermined cause. In an in vitro experiment, monoclonal anti-A5-antibodies also prevented the formation of the syncytiotrophoblast, which is a possible cause of early foetal loss. However in the above-mentioned study [10], only 4 out of 8 women with unexplained pregnancy losses tested positive for anti-A5 antibodies. Another study has also failed to demonstrate a significant correlation between these antibodies and pregnancy loss. Testing for anti-A5antibodies should therefore only be recommended in patients suffering from thromboembolic events when tests for phospholipid antibodies are negative. Summary Testing for antibodies associated with APS is only indicated if the test results are likely to lead to beneficial therapeutic consequences. As long as the benefit of prophylactic therapy in individuals with antibodies but without clinical symptoms remains unproven, tests should only be performed in patients whose symptoms are typical of APS or whose symptoms cause the condition to be suspected [Table 3]. As an initial strategy the established and well standardised tests for lupus anticoagulants and ß2-GI-dependent cardiolipin antibodies should be performed. If these tests are negative in individuals with clinical APS symptoms, we recommend testing for antibodies against other phospholipids and proteins as shown in Table 4. When interpreting the results from the tests described above, it should always be remembered that these test systems are so far not standardised and that the clinical utility of the tests has not yet been proven in large patient cohorts. In the individual patient, however, the detection of one of these antibodies can be very helpful in deciding the optimal course of further therapy. References 1. Tsatsumi A, Koike T. Measurement of Anti-ß2-glycoprotein I Antibodies. In: Khamashta MA (Editor). Hughes Syndrome: Antiphospholipid Syndrome. Springer 2000; 238-244. 2. Harris EN. The second international anticardiolipin standardization workshop/the Kingston Table 4. Recommended antibody testing in individuals suspected of having APS. Stroke 1998; 29: 1759-1764. 8. Woehrle R, Oppermann M, Matthias T, Helmke K. Clinical relevance of antibodies against different phospholipids. Abstract book of the Third European Forum on Antiphospholipid Antibodies held in Utrecht, April 20th - 21st, 2001; 70. 9. Miesbach W, Matthias T, Scharrer I. Identification of thrombin antibodies in patients with antiphospholipid syndrome. Ann N Y Acad Sci 2005; 1050: 250-256. 10. Woehrle R, Oppermann M, Matthias T, Helmke K. Pregnancy losses are stronger correlated with antibodies against thrombin than with other phospholipid- or protein-antibodies. Autoimmunity reviews 2004; 3 Supplement 2: 44. The authors Dr Rainer Woehrle and Prof. Dr Klaus Helmke Dept. for Rheumatology and Clinical Immunology, Hospital München-Bogenhausen, Englschalkinger Str. 77, D-81925 München, Germany