Anticardiolipin (aCL) tests are important to aid in the diagnosis of antiphospholipid syndrome (APS). 1 Patients affected with APS are subject to episodes of thrombosis in arteries and/or veins, pregnancy loss (probably secondary to thrombosis of vessels in the placenta), and thrombocytopenia, associated with antiphospholipid (aPL) antibodies. 1,2 Antiphospholipid antibodies are autoantibodies directed against anionic phospholipids or protein-phospholipid complexes measured in solid-phase immunoassays as aCL or detected in phospholipid-dependent clotting tests as lupus anticoagulant (LA). 3-5 The diagnosis of APS is based on finding a moderate-to-high positive aCL test and/or an LA test with any one of the characteristic clinical features presented previously. 2
Although a sensitive test, the standard aCL enzyme-linked immunosorbent assay (ELISA) may be positive in a variety of disorders, including connective tissue diseases, infectious disorders such as syphilis, Q fever, and AIDS, and in some drug-induced disorders. 6-10 It generally is believed that aCL antibodies are clinically significant only when present in APS; thus, there have been numerous efforts to modify the aCL ELISA to make it more specific for APS. In addition, based on an early observation that patients with high-positive IgG aCL results were more likely to have APS 11 and that aCL are heterogenous in function and specificity and measured using a wide variety of techniques, substantial efforts have been devoted to quantifying the aCL ELISA test in a standardized manner. 12-15 This chapter discusses the various techniques used in the diagnosis of APS, the results of aCL workshops that have attempted to validate and improve measurement of aCL antibodies, and issues related to problems and solutions in aCL testing. An overview of new and more specific tests for diagnosis of APS is also included.
History of the Anticardiolipin Test
In 1983, a group of investigators at the Hammersmith Hospital in London, England noticed that some patients with systemic lupus erythematosus (SLE) had a rather uncommon coagulation abnormality, LA, that was characterized by the abnormal prolongation of the partial thromboplastin time (PTT). 16 The investigators also established that instead of abnormal bleeding, these patients were subject to thrombosis. 16 Soon thereafter, the LA phenomenon was shown to be caused by an autoantibody believed to bind phospholipids because it inhibited two phospholipid-dependent coagulation reactions in the clotting-cascade: the prothrombin-thrombin conversion and the activation of factor X. In addition, approximately 25-50% of patients with the LA reaction also had a biologically false-positive test for syphilis (BFP-STS). Antibodies responsible for the BFP-STS were known to bind cardiolipin, a negatively charged phospholipid. The LA test had some drawbacks: it was a functional assay affected by a number of variables, including preparation and storage of samples and the type of reagent used. In addition, the test lacked sensitivity and could not be readily standardized. Thus, this group of investigators reasoned that use of a solid-phase immunoassay with cardiolipin as antigen might be one way of detecting antibodies with LA activity. 17 They also reasoned that such a test would have the advantages of possibility of using stored serum, greater sensitivity, more reproducibility, better quantitation, and the possibility of standardization. The group succeeded in establishing a solid-phase radioimmunoassay with cardiolipin as antigen, and the antibodies detected were termed aCL. Hence, the first anticardiolipin test was established in 1983. 17 The assay then was converted to ELISA that was safer than radioimmunoassay and easier to perform. 18 The test proved more sensitive than the LA assay and enabled diagnosis of a much larger number of patients with APS. The aCL antibodies were found to cross-react with negatively charged phospholipids, such as phosphatidylserine (PS) and phosphatidylglycerol. 19,20 Thus, the name aCL antibodies was changed to aPL, and the disorder with which these antibodies were associated was called APS. 1,2,20
Widespread adoption of the solid phase anticardiolipin assay led to several potential problems. These antibodies were soon reported in several disorders such as syphilis, AIDS, and connective tissue diseases, as well as in healthy individuals who did not have the features of the disorder. 6,7,9 False-positive tests in the aforementioned conditions could be best explained by the sensitivity of the aCL test. Methods of performing the test also varied, however, and results were questionable in some instances. Fortunately, it was recognized that the majority of patients with APS tended to have high aCL antibody levels, usually of the IgG isotype. To ensure that the aCL test would retain its value in diagnosis of APS, it would be necessary to identify antibodies by isotype and to quantify results using some reliable unit of measurement. There also was a need to establish which testing methods were valid, as well as standard procedures for performing the solid-phase immunoassay. To achieve these goals, an international standardization workshop was first conducted in 1986, and subsequently other workshops were held. 12
Tests Used in Diagnosis of Antiphospholipid Syndrome
ANTICARDIOLIPIN TEST
Several changes and improvements have been made to the first aCL test to reduce the background binding, to quantitate the results in units, and to optimize the incubation times and temperatures. 12-15,18 The introduction of adult bovine or fetal calf serum was found to enhance antibody binding to cardiolipin, an effect later attributed to a serum protein termed β2glycoprotein 1 (β2GP1). The current aCL test is sensitive and is positive in more than 80-90% of patients with APS. An association of a positive aCL test with clinical manifestations of APS occurs principally with persistent medium-to-high levels of aCL (or aPL) antibodies, and the IgG isotype is more prevalent in APS patients than isolated IgM. 18 There are reports, however, of isolated IgM aCL, and occasionally isolated IgA aCL antibodies, associated with clinical manifestations of APS. 20-22 IgA aCL antibodies have been shown to have pathogenic properties in an animal model of thrombosis, but studies show that the detection of IgA aCL antibodies does not provide additional help in diagnosing APS. 23 O'Callaghan et al 21 studied 795 patients classified into five different groups with various manifestations of APS and SLE and found that significant levels of IgA aCL antibodies were present in only two patients. In agreement with that study and in our experience, the presence of IgA aCL antibodies (with a few exceptions) is accompanied by a positive IgG and/or IgM aCL test in patients with APS.
Interlaboratory variation has plagued the study of APS since its earliest recognition as a distinct disorder. It is now established that interlaboratory agreement is best when positive aCL results are reported in semiquantitative terms (ie, low-positive, medium-positive, or high-positive). 13 Even then, reasonable reproducibility and acceptable clinical correlation require standardization of the test. Because of these concerns, there has been considerable effort directed toward standardizing aPL test procedures and defining antibody levels. 12-15
Standardization of the Anticardiolipin Test
In the first anticardiolipin workshop, the assay methods that enabled valid measurements of aCL antibody levels were determined. 12 In addition, units of measurement were established, and six calibrator standards were introduced to assist laboratories worldwide in establishing the aCL assay. The second workshop demonstrated that semiquantitative measures of aCL antibody levels enabled the best agreement between laboratories. 13 The third and fourth workshops sought to settle controversial issues about aCL specificity and to critically examine some of the newly introduced commercial kits. 14,15 Together, these workshops have demonstrated that investigators working collaboratively can improve testing methods and enhance the understanding of basic scientific issues such as those related to aCL specificity. Despite these efforts in standardization, a considerable degree of interlaboratory variation still exists. 24-26
National and international organizations are contributing to the standardization of the aCL test: the College of American Pathologists enrolls certified laboratories in quality control surveys for aCL testing and requires participation in the program for accreditation purposes. In addition, the College attempts to improve agreement between laboratories by distributing aCL survey samples and evaluating results. The National Committee for Clinical Laboratory Standards will soon start distributing the first proposed guidelines for the determination of aCL antibodies.
Testing for Antibodies to Other Phospholipids
The use of other antigens used to coat the ELISA plates has been reported for the detection of aPL antibodies. 20 A number of laboratories include in their test panel assays to detect antibodies directed to other negatively charged phospholipids such as phosphatidylserine, phosphatidic acid, phosphatidylinositol, or phosphatidylglycerol. Some reports indicated that APS patients may have antibodies to zwitterionic phospholipids (such as phosphatidylethanolamine) or to positively charged phospholipids such as phosphatidylcholine. 27,28 The value of these tests is still uncertain because no standardized procedure has been established and calibrators are not available. In general, based on the existing clinical and serologic evidence, these tests do not provide additional help in identifying patients with APS.
LUPUS ANTICOAGULANT
The LA is less frequently positive in APS and is a more specific test for detection of aPL because the LA reaction is found much less frequently in non-APS disorders. 29 The LA test (or, rather, sequence of tests) measures the ability of aPL autoantibodies to prolong phospholipid-dependent clotting reactions. Although aPL antibodies detected in both the aCL and LA tests are specific for phospholipids, the involved binding proteins or a complex of molecules are heterogenous, and the two tests (aCL and LA) do not necessarily identify the same antibodies. 30 Further, although the majority of patients with APS have positive aCL and LA tests, approximately 10-16% of them are positive for LA and negative for aCL, and 25% are positive for aCL and negative for LA.
More Specific Tests for APS
Although the aCL ELISA is a sensitive test, one of its major drawbacks is that it may be positive in a number of disorders other than APS. Anticardiolipin antibodies have been reported in numerous infectious diseases, including syphilis, AIDS, hepatitis C, Q fever, tuberculosis, parvovirus B19 infections, and cytomegalovirus, and can be induced by medications. 6-10,31-33 Because patients with APS usually have higher aCL levels, greater specificity in diagnosis has been achieved by the use of higher cut-off points. Alternatively, newer assays that use phosphatidylserine, 19 a mixture of negatively charged phospholipids (APhL ELISA Kit; Louisville APL Diagnostics, Doraville, GA), 34,35 or β2GP1 have been proposed for more specific measurements of antibodies present in APS. 36-38 Some reports now suggest that both the aCL ELISA and a more specific test be performed for patients suspected of having APS. 39
The new more specific assay must also be sensitive if a confident diagnosis of APS is to be made. Three antigen preparations are likely candidates. The first and most extensively studied antigen is β2GP1. 34-36 The second and third are the APhL phospholipid mixture used as the antigen in an ELISA Kit (APhL ELISA Kit; Louisville APL Diagnostics) 34,35 and phosphatidylserine, available in several commercial assays.
Current studies show that β2GP1, particularly when coated on oxidized or high-binding polystyrene ELISA plates, is a relatively specific antigen for autoantibodies present in APS patients. 36 Antiβ2GP1 antibodies have been reported to be associated primarily with thrombosis in patients with APS, but studies also have shown these antibodies in patients with pregnancy loss and other manifestations of APS. 40-43
Various methods have been reported for the detection of antiβ2GP1 antibodies, 36,37,44 but only one study reported the adoption of units of measurement and establishment of ranges of positivity, cut-off levels, and intraassay and interassay variations. 45 Studies from several laboratories suggest that the sensitivity of the antiβ2GP1 test for APS varies from 40-90%. 40,46,47 It remains uncertain, however, whether the antiβ2GP1 test is truly specific for APS in that it is positive in some infections. 48-50
The second antigen is the APhL phospholipid mixture prepared as a kit by Louisville APL Diagnostics. (APhL ELISA Kit). Two of the authors of this review were principals (ENH and SP) in developing both the antigen and the kit, and evaluation of data presented herein should take this into account. The APhL phospholipid mixture was determined on the basis of testing of aCL-positive sera from a large number of patients with and without APS. Negatively charged phospholipids were tested singly and as mixtures to determine which would best distinguish APS sera from aCL-positive non-APS samples. A mixture of phospholipids was identified that enabled such distinction while retaining sensitivity for detection of APS. Four published studies have examined this antigen. 15,34,35,51 The largest included 438 patients with various connective tissue diseases, 33 patients with APS, and 200 healthy control subjects, and sera were examined using the standard aCL ELISA and the APhL ELISA Kit (Louisville APL Diagnostics). The sera were prepared and labeled in one center and tested blindly in another laboratory; and results were analyzed in the center where the samples were labeled. All patients with APS had positive tests for aCL antibodies, and 30 of 33 tests were positive using the APhL ELISA Kit (90.9% sensitivity; Louisville APL Diagnostics). In patients without APS, 45 of 438 tested positive for aCL, but only 9 of 438 sera were positive in the APhL ELISA Kit (99.5% specific; Louisville APL Diagnostics). These data suggest that the APhL ELISA Kit (Louisville APL Diagnostics) may be a sensitive and relatively specific means of identifying patients with APS. 34,35,51
In another study of 54 APS samples, comparisons were made using the aCL ELISA, the APhL ELISA Kit (Louisville APL Diagnostics), and the antiβ2GP1 ELISA. The sensitivity for the antiβ2GP1 assay was 74% (Table 1). The specificity of the antiβ2GP1 varies among groups of investigators and publications, however, depending on the selection patient sera and the technique used. 46-50,52 In a series of experiments performed by our group, in 184 samples from non-APS patients who had syphilis or other autoimmune disorders, 18 of 184 samples were positive, yielding a specificity of 82% (Table 2), considerably better than the specificity of the aCL ELISA. Most other investigators agree that the antiβ2GP1 test is less sensitive but more specific than the aCL ELISA for diagnosis of APS. In the same series of experiments, the APhL ELISA Kit (Louisville APL Diagnostics) was 98% sensitive and 99% specific.
In another recent study, 69 samples from patients with syphilis, 30 with leishmaniasis, and 33 with leptospirosis were tested with the aCL ELISA, the antiβ2GP1 ELISA, and the APhL ELISA Kit (Louisville APL Diagnostics). The results, shown in Table 3, indicated that the APhL ELISA Kit (Louisville APL Diagnostics) was more specific than either of the other two assays. Interestingly, 36 samples of the 132 samples tested in this study were also positive in the antiβ2GP1 ELISA, yielding a specificity of only 70%. This relatively high incidence of antiβ2GP1 antibodies in the sera in this group of infectious diseases is not understood and requires further investigation.
In September 1996, a fourth anticardiolipin workshop was conducted. 15 The objective of this most recent workshop was to compare four techniques for measurement of aCL antibodies to determine which was more specific for identification of APS. Samples tested were eight APS, four aCL-positive syphilis, two aCL-positive (one obtained from a patient with Q fever and the other from a patient with SLE, neither of whom had features of APS), and six from normal healthy control subjects. Tests evaluated were the standard aCL ELISA, two commercial aCL ELISA kits (INOVA Diagnostics, San Diego, CA and Incstar, Stillwater, MN), the APhL ELISA Kit, an antiβ2GP1 kit (INOVA Diagnostics) and a flow cytometric technique in which IgG and IgM antibodies to cardiolipin and to phosphatidylserine can be determined simultaneously.
All techniques tested were 90-100% sensitive, correctly identifying almost all sera from patients with APS (Table 4). Results varied, however, with respect to specificity. The APhL ELISA Kit (INOVA Diagnostics) was most specific (100%), and the antiβ2GP1 assay was nearly as specific, with only the Q fever sample (but not the syphilis or SLE samples) falsely identified as positive. The flow cytometric assay method with phosphatidylserine as antigen showed the third best specificity, falsely identifying only two samples. None of the aCL assays proved to be 100% specific. The syphilis, Q fever, and SLE samples were all reported as positive. 15
Finally, there has been an increasing number of reports from various centers suggesting that patients with APS have antibodies that bind proteins other than β2GP1, including prothrombin, 53 protein-C, protein-S, 54 and annexins. 55 There are too few reports at this time from which to clearly establish the diagnostic value of these assays. Determination of the value of ELISA tests using protein antigens other than β2GP1 will require validation, standardization of the techniques, and testing of large numbers of sera samples from patients with APS as well as with other disorders to determine their diagnostic and predictive values.
What Test Should Be Used for Diagnosis of Antiphospholipid Antibodies
Correct identification of patients with APS is important, because prophylactic anticoagulant therapy can prevent thrombosis from recurring, and treatment of affected women during pregnancy can improve fetal and maternal outcome. But there are numerous causes of thrombosis and of pregnancy loss, and properly diagnosing APS requires a positive LA or aPL test. The LA and aCL ELISA generally are accepted confirmatory tests for APS. Recently, a forum of antiphospholipid experts gathered at a special session during the eighth International Symposium on Antiphospholipid Antibodies agreed that these two tests should be used primarily in the diagnosis of APS. 56 They concluded that a diagnosis of APS can be made with confidence in patients who have well-documented clinical features and a moderate-to-high positive aCL antibody or LA test result. There are a number of situations in which the antiβ2GP1 or the APhL ELISA Kit (Louisville APL Diagnostics) might be used to confirm diagnosis of the APS. These include: patients with venous or arterial thrombosis or with pregnancy loss who are low-positive for IgG aCL, have only IgM or IgA aCL, or who have equivocal features of APS. Additional examples include patients with idiopathic thrombocytopenia, atherosclerosis, first trimester pregnancy losses, or instances in which venous or arterial thrombosis or recurrent pregnancy loss may be attributable to factors other than APS. Unusual presentations of APS, such as chorea, transverse myelopathy, livedo reticularis, leg ulcers, or cardiac valvular lesions in the absence of pregnancy loss or thrombosis also may benefit from tests of proven specificity.
Based on the knowledge and experience accumulated to date, we propose a stepwise approach to the laboratory diagnosis of APS. In our algorithm, aCL and LA testing should be performed as first-line tests. If these results are negative or equivocal, more specific tests, such as antiβ2GPI or the APhL ELISA Kit (Louisville APL Diagnostics), should be performed.
Summary
The first aCL test was developed in 1983 and subsequently standardized. Although new and more specific tests have become available, the aCL ELISA and the LA tests are still the first choice to be used in diagnosis of APS. Newer tests such as the antiβ2GP1 ELISA and the APhL ELISA Kit (Louisville APL Diagnostics) use somewhat different antigens and likely provide a more specific (and possibly more reliable) diagnosis of APS while retaining good-to-excellent sensitivity. Other tests, such as ELISA for prothrombin antibodies and annexin V antibodies, are still undergoing development and will require standardization and extensive evaluation.
We thank Dr Isabel Abreu and Dr Mittermeyer B. Santiago for performing some of the studies reported in this review.
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