Autoantibodies have been implicated in a variety of diseases.

Antiphospholipid Antibodies Douglas A. Triplett, MD Objective. To review the role of lupus anticoagulants in the pathogenesis of both venous and arterial thromboembolic events, as well as in recurrent spontaneous abortions. The pathophysiology of lupus anticoagulants and associated antiphospholipid antibodies (eg, anticardiolipin antibodies) is also discussed. Data Sources. Review of the recent medical literature. Data Extraction and Synthesis. Key articles in the recent medical literature dealing with lupus anticoagulants and their role in pathogenesis of thromboembolic events were reviewed. Plasma proteins that have an affinity for binding to perturbed cellular membranes have been identified as the antigenic targets for antiphospholipid antibodies. Thus, the concept of antiphospholipid antibodies needs to be reevaluated. Perhaps a better term is antiprotein-phospholipid antibodies. The principal antigenic protein targets are 2 -glycoprotein I, prothrombin, and a wide range of additional proteins that interact with activated cellular membranes, including protein C, protein S, annexin V, etc. Most research reported in the literature has focused on 2 -glycoprotein I and human prothrombin. (Arch Pathol Lab Med. 2002;126:1424 1429) Autoantibodies have been implicated in a variety of diseases. The diversity of these antibodies has stimulated multidisciplinary interest in linking these antibodies to potential pathophysiologic mechanisms. Antiphospholipid antibodies (APAs) were first described by Wassermann et al in 1906. 1 The Wassermann test was a complement fixation procedure using saline extracts from the liver of fetuses with congenital syphilis. Following the introduction of the Wassermann test, a variety of procedures using antigens derived from alcoholic extracts of normal tissue were described. These tests used either complement fixation (Kahn) or flocculation techniques (Venereal Disease Research Laboratory [VDRL], rapid plasma reagin test, and automated reagin test). Pangborn 2 was the first to identify an acidic phospholipid as the antigen present in alcohol extracts of bovine heart. This extract was subsequently named cardiolipin. The antigen used in the VDRL test is a mixture of cardiolipin-lecithin-cholesterol. False-positive tests using the VDRL were classified as either acute or chronic biologic false positives (BFPs). 3 The chronic BFP was defined as a positive nontreponemalbased screening test that persisted for 6 months or longer. In many cases, patients with chronic BFP serologic tests for syphilis (BFP-STS) were found to have underlying autoimmune disease, including systemic lupus erythematosus (SLE), rheumatoid arthritis, and Sjögren syndrome. 4 Chronic BFP-STS may be the first laboratory finding of an Accepted for publication April 8, 2002. From the Department of Pathology, Indiana University School of Medicine; Midwest Hemostasis and Thrombosis Laboratories, and Department of Pathology, Ball Memorial Hospital, Muncie, Ind. Presented at the College of American Pathologists Consensus Conference XXXVI: Diagnostic Issues in Thrombophilia, Atlanta, Ga, November 9 11, 2001. Reprints: Douglas A. Triplett, MD, Department of Pathology, Ball Memorial Hospital, 2401 West University Ave, Muncie, IN 47303-3499 (e-mail: dtriplett@chs.cami3.com). autoimmune disease and often precedes the onset of clinical symptoms. An unusual circulating anticoagulant was first described by Conley and Hartmann in 1952. 5 The title of the article suggested the patients had a hemorrhagic disorder; however, no documentation of significant clinical bleeding was described. Subsequently, Laurell and Nilsson 6 reported an association between BFP-Wassermann test and a circulating anticoagulant. These investigators also noted the anticoagulant could be adsorbed by the Kahn reagent, which was used in syphilis testing. Laurell and Nilsson were the first to link the anticoagulant to an antibody that reacted with a lipid antigen. Subsequently, in 1972, Feinstein and Rapaport 7 proposed the term lupus anticoagulant (LA) for these antibodies. Lupus anticoagulant is a misnomer, since the vast majority of patients with laboratory findings of LA do not have SLE. 8 Harris and colleagues 9 at St Thomas Hospital in London, England, were the first to report a sensitive test system to identify anticardiolipin antibodies (ACAs). In contrast to earlier studies, they used a solid-phase radioimmunoassay, which was 200 to 400 times more sensitive than the VDRL. They found 62% positivity among 65 patients with SLE or other autoimmune diseases. There was also a strong correlation of positive ACA results and LA (29 [91%] of 32 patients) and thromboembolic events. ANTIPHOSPHOLIPID ANTIBODIES Immunology The term antiphospholipid antibodies is a misnomer in the majority of patients presenting with clinical and laboratory findings consistent with antiphospholipid antibody syndrome (APS). 10 There are 2 distinct groups of antibodies, some of which react only with phospholipids (true antibodies to phospholipids, in most cases infection-induced). These antibodies are usually transient and disappear following convalescence. 11 In the case of patients who have APAs in the setting of 1424 Arch Pathol Lab Med Vol 126, November 2002 Antiphospholipid Antibodies Triplett

Table 1. Antiphospholipid Antibodies; Protein Targets 2 -Glycoprotein I Annexin V Thrombomodulin Factor XI High-molecular-weight kininogen Factor XII Prothrombin Protein C Protein S Activated protein C Complement factor H Low-molecular-weight kininogen autoimmune diseases (eg, SLE, primary APS, drug-induced APAs), a variety of plasma protein antigenic targets have been identified (Table 1). These autoantibodies recognize plasma proteins that bind to activated cellular membranes (eg, monocytes, endothelial cells, platelets). 12,13 Among the proteins binding to activated cellular surfaces are 2 -glycoprotein I ( 2 -GPI), prothrombin, annexin V, protein C, protein S, and proteins from the kininogen system (high- and low-molecular-weight kininogens). 13 An important aspect of the pathophysiology is the density of proteins binding to injured cellular surfaces. Increased density of plasma proteins allows for bivalent bonding of the antibodies, which in turn enhance the prothrombotic nature of the injured cells. 14 Antigenic Targets 2 -Glycoprotein I (Apolipoprotein H). 2 -Glycoprotein I is a member of the short-consensus-repeat protein family. It is a 50-kd glycoprotein found in plasma with a concentration of approximately 200 g/ml. 15 2 -Glycoprotein I is characterized by 5 sushi domains. The fifth sushi domain contains the binding site, which attaches to activated cellular surfaces. 16 Lysine-rich segments are found in the fifth sushi domain and are responsible for binding to activated cellular surfaces. The physiologic role of 2 -GPI remains somewhat enigmatic. In vitro studies have found 2 -GPI inhibits prothrombinase activity, the contact system of blood coagulation, and adenosine 5 diphosphate induced platelet aggregation. 17 2 -Glycoprotein I may also play a role in clearance of apoptotic cells. 18 Patients who have been described with an absence of 2 - GPI have not experienced an increased risk of thrombosis. Antiphospholipid antibodies bind to 2 -GPI primarily in the third and fourth sushi domains. 18 However, there is some controversy. A research group recently has proposed sushi domain I as the principal target of autoantibodies with pathogenic consequences. 19 Lupus anticoagulants have been shown to enhance binding of 2 -GPI to activated phospholipid cellular surfaces. Prothrombin. Prothrombin is also a common antigenic target for APAs. 20 These antibodies bind to prothrombin, prethrombin I, and fragment I but not to thrombin. Antibodies to prothrombin lead to prothrombin clustering (antigenic density) on surfaces of activated cells. Antibodies to prothrombin may account for approximately two thirds of LAs identified in patients with APS. Although these antibodies tend to have low affinity, there are occasions when high-affinity antibodies to prothrombin lead to hypoprothrombinemia. 21,22 Often these cases are seen in children with associated hemorrhagic complications. 23 These antibodies have species specificity for human prothrombin. Antibodies to prothrombin have also been described in adults. In many cases, these antibodies are detected in young women with evidence of autoimmune disease (eg, SLE, rheumatoid arthritis). Recently, commercial enzyme-linked immunosorbent assays (ELISAs) for detection of antibodies to prothrombin have been approved for clinical testing. Levels of calcium in the test system are important because they affect the confirmation of prothrombin. Antibodies to Oxidized Low-Density Lipoprotein. The association of APAs and oxidized low-density lipoprotein has recently been reported. Typically, the antibodies are 2 -GPI dependent. In a study of patients with SLE and APA positivity, increased quantities of isoprostanes were found in urine specimens. 24 The isoprostanes reflect in vivo lipid peroxidation. Treating these patients with vitamin E resulted in significant decreases in urinary isoprostanes. Animal models using mice or other animals have found that immunization with 2 -GPI or other antigens induces in vivo thrombosis. 25 In addition, human antibodies injected into animals also promote thrombosis. Clinical Complications The range of clinical complications reported with LA/ ACA involves virtually every organ system. 26 Antiphospholipid antibody is one of the most common causes of acquired thrombophilia. Arterial thrombotic events are reported most frequently in the cerebral circulation. 27 Approximately one third of strokes in patients younger than 50 years have been attributed to APAs. 28 On the venous side of the circulation, an estimated 15% of deep vein thromboses (DVTs) are due to the presence of APAs. 29 In addition, a number of other venous sites have been associated with thromboembolic events, including mesenteric, renal, and adrenal veins. Clinically, catastrophic APS (CAPS) may resemble thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. 30 Recurrent thromboembolic events tend to have consistency with respect to site (ie, arterial-arterial, venous-venous). A number of obstetrical complications have been linked to APAs, including maternal DVT, chorea, and eclampsia/ preeclampsia. Recurrent spontaneous abortions have also been linked to APAs. 31 Between 5% and 15% of all recurrent spontaneous abortions have been attributed to APAs. Virtually every clinician will encounter patients with APAs. Neurologic complications include chorea, stroke, migraine, and multi-infarct dementia. 32 Dermatologists not infrequently are presented with patients with livedo reticularis and, more uncommonly, ulceration or digital gangrene. 33 In patients with stroke, it is important to suspect an underlying presence of APAs. 27,28 Patients with APAs may have a Libman-Sacks endocarditis. In many cases of young adults with stroke linked to APA, mitral or aortic valves are the origin of the emboli. 34 In APA-positive patients with a history of cerebral events, transesophageal echocardiography should be a standard part of the evaluation. In the early 1990s, an accelerated form of thrombosis affecting the microvasculature of many organs was identified and linked to APAs. 35 Clinically, these patients had multiorgan thrombosis. Precipitating factors initiating CAPS included medications, minor and major surgical procedures, and in some cases, infections. The underlying pathophysiology remains a matter of conjecture. Throm- Arch Pathol Lab Med Vol 126, November 2002 Antiphospholipid Antibodies Triplett 1425

Thrombotic mechanisms Antigenic target(s) Table 2. Catastrophic Antiphospholipid Syndrome: Differential Diagnosis CAPS TTP HIT DIC Antibody-mediated thrombosis (acquired) Annexin V, 2 -glycoprotein I, metalloprotease (?), prothrombin Antibody-mediated thrombosis/or absence of enzyme Metalloprotease that cleaves von Willebrand factor Antibodies to platelet factor 4, IL-6, etc, when complexed with heparin Platelet factor 4 heparin Anatomic site of thrombosis Microvasculature Microvasculature Venous and arterial, microvasculature (unusual) Schistocytes Rarely Platelets and coagulation factors Acute Infections with septicemia, malignancies, drug-induced, cirrhosis, etc. NA Microvasculature, venous and arterial Platelet consumption Platelet consumption Platelet consumption Consumption of coagulation factors VIII, XIII, and I and platelets Fibrin Degradation Products APA (LA and ACA) * APAs indicates antiphospholipid antibodies; LA, lupus anticoagulant; ACAs, anticardiolipin antibodies; CAPS, catastrophic antiphospholipid syndrome; TTP, thrombotic thrombocytopenic purpura; HIT, heparin-induced thrombocytopenia; DIC, disseminated intravascular coagulation; NA, not available;, present;, absent; and, may be present or absent in a given patient. Table 3. Clinical Venous thrombosis Arterial thrombosis Recurrent fetal loss Thrombocytopenia Criteria for Antiphospholipid Antibody Syndrome* Laboratory IgG anticardiolipin antibody ( 10 GPL units) Positive lupus anticoagulant test IgM anticardiolipin antibody ( 10 MPL units) and positive lupus anticoagulant test * Taken from Harris and Bos. 35 Patients with the antiphospholipid antibody (APA) syndrome should have at least 1 clinical and 1 laboratory finding during their disease. The APA test(s) should be positive on at least 2 occasions more than 8 weeks apart. The diagnosis of lupus anticoagulant should be confirmed using the criteria established by the Scientific and Standardization Subcommittee for Standardization of Lupus Anticoagulants. 38 GPL and MPL refer to IgG and IgM phospholipid antibodies. The units refer to the standards proposed by Harris and Bos 35 and the Second International Workshop on Phospholipid Antibodies, April 4, 1986. botic involvement of the microvasculature/capillaries is a consistent finding in patients with CAPS. Endothelial cell activation appears to be a part of the pathophysiologic process. 30 The differential diagnosis of CAPS includes thrombotic thrombocytopenic purpura, disseminated intravascular coagulation, and hereditary thrombophilia (eg, factor V Leiden, prothrombin G20210A, protein C deficiency, protein S deficiency, antithrombin deficiency). Table 2 summarizes the antibody-mediated thrombotic conditions. ANTIPHOSPHOLIPID ANTIBODY SYNDROME History and Definition Hughes and colleagues (St Thomas Hospital/London) initially suggested the term anticardiolipin syndrome to define the association of positive ACA results and clinical findings. 36 Table 3 summarizes the criteria necessary to establish the diagnosis of APS. To establish the diagnosis of APS, the patient must have at least 1 of the major clinical findings (venous thrombosis, arterial thrombosis, recurrent fetal loss, and thrombocytopenia). In addition, there should be a positive laboratory test result indicating the presence of either ACAs or positive LA testing (eg, prolonged dilute Russell viper venom test [drvvt], prolonged activated partial thromboplastin time [aptt]). An important caveat with respect to application of the guidelines to identify APS is the need to retest the patient approximately 8 to 10 weeks following the initial positive laboratory results. This step is important in identifying transient APAs, which often are the result of intercurrent infections. Infection-related APAs are not associated with thromboembolic events or other complications (recurrent spontaneous abortion, livedo reticularis, neurologic findings, etc). LABORATORY IDENTIFICATION OF LUPUS ANTICOAGULANTS The appropriate collection and preparation of plateletpoor plasma (PPP) is an often-overlooked initial step in coagulation testing. A traumatic venipuncture may significantly compromise the blood sample. Separation of PPP from the cellular elements of blood should be done expeditiously. Optimally, the number of residual platelets in the PPP should be less than 5000/ L. In many situations, the PPP may be frozen ( 80 C) and tested at a later date. If there are significant residual platelets in PPP, one may see a platelet neutralization effect on thawing PPP for LA testing. 37 According to the guidelines established by the Scientific Subcommittee for Lupus Anticoagulants/Phospholipid- Dependent Antibodies, screening coagulation testing must use more than 1 test system (eg, aptt, dilute prothrombin time, drvvt). 38 Currently, at least 2 of the tests listed are recommended; however, many laboratories choose to do all 3 of these tests. Other tests that have been used include Textarin Time and Taipan Venom Test (Table 4). The Kaolin Clotting Time has also been used as a screening procedure. This test system is exquisitely sensitive to residual platelets that may compromise the test results. The drvvt has become a very popular screening test in both the United States and Europe. Thiagarajan et al 39 were the first authors to describe the use of a modified RVVT for the diagnosis of LA. Their studies found the drvvt to be more sensitive than aptts and the tissue thromboplastin inhibition test (synonym: dilute prothrom- 1426 Arch Pathol Lab Med Vol 126, November 2002 Antiphospholipid Antibodies Triplett

Table 4. Rusell viper venom Textarin Taipan Venom Lupus Anticoagulants: Venom-Based Assays (Variables)* Factor X No No Factor V No Factor II Ca 2 Platelets * This table summarizes data on snake venom based assays that have been used to identify the presence of lupus anticoagulants. Russell viper venom is the most commonly used venom-based assay. The diluted Russell viper venom test (drvvt) has been used as a screening assay. A second drvvt reagent that has added phospholipids was developed as a confirmatory assay. Antibodies to factors X, V, and II may give a prolonged drvvt result. Textarin is a reagent obtained from a venomous Australian snake, Pseudonaja textalis. In contrast to Russell viper venom, Textarin is not sensitive to abnormalities involving factor X. Taipan Venom assesses the conversion step of prothrombin to thrombin. The Taipan Venom Time is insensitive to abnormalities of factor X or factor V. Table 5. Laboratory Diagnosis of Lupus Anticoagulants 1. Demonstration of an abnormal phospholipid-dependent screening test of hemostasis (eg, aptt, drvvt, KCT, dpt, Textarin Time, Taipan Time) 2. Failure to correct the prolonged screening coagulation tests on mixing with normal platelet-poor plasma (criteria necessary to identify the presence of a circulating anticoagulant [synonym: inhibitor]) 3. Shortening or correction of the prolonged screening tests on the addition of excess phospholipids or hexagonal-phase phospholipids 4. Rule out other coagulopathies (eg, factor VIII inhibitors, presence of heparin) * appt indicates activated partial thromboplastin time; drvvt, dilute Russell viper venom test; KCT, Kaolin Clotting Time; and dpt, dilute prothrombin time. bin time). There are a number of commercially available drvvt test systems. However, there is variability among manufacturers. 40 When choosing a drvvt test system, laboratories must carefully identify the reagent with the best sensitivity and specificity for LA detection. Table 5 summarizes the recommendations of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis: Subcommittee for Lupus Anticoagulants/Antiphospholipid Antibodies. 38 In response to the recommendations cited, commercial reagent manufacturers have provided confirmatory reagents using a drvvt test with excess phospholipid. These reagents fulfill the criterion to shorten or correct the prolonged baseline drvvt. The same principle of an initial screening reagent with a low phospholipid concentration and a confirmatory reagent with higher phospholipid concentration applies to the other tests used as initial screens to detect a circulating anticoagulant (aptt, dilute prothrombin time, etc). In addition to the integrated drvvt test systems, Diagnostica Stago developed the Staclot LA test. 41 This system uses a 2-step approach to establishing LA diagnosis. The Figure illustrates the sequential steps involved in the Staclot LA procedure. The test uses hexagonalphase phospholipid to neutralize an LA, which results in shortening of the clotting time in tube 2. The assay system Summary of the Staclot LA procedure. Diluent is added to tube 1 and egg phosphatidylethanolamine (EPE) to tube 2. Subsequently, a source of normal platelet poor plasma with polybrene (to neutralize heparin in patient samples) is added to both tubes followed by incubation and the addition of an aptt reagent. In the presence of a LA, there will be significant shortening of the aptt result in Tube 2. A difference of greater than 12 seconds between Tube 1 and 2 is considered to be a positive test for LAs. 41 Arch Pathol Lab Med Vol 126, November 2002 Antiphospholipid Antibodies Triplett 1427

also contains a heparin neutralizer, which enhances the specificity of Staclot LA. Another approach to confirm the presence of LA is the Platelet Neutralization Procedure. 37 This is a simple test using washed frozen platelets. An aliquot of the freezethawed platelets is added to the patient plasma, and a second aptt is determined. Addition of the freeze-thawed platelets to LA-positive PPP results in significant shortening of the prolonged baseline value. The test is simple to perform and has relatively high specificity. To correct for the dilutional effect of the freeze-thawed platelets, a second mixture of patient plasma together with buffered saline is used as a control. According to the guidelines established by the Scientific and Standardization Committee Subcommittee on Lupus Anticoagulants/Phospholipid-Dependent Antibodies, 2 or more LA screening tests are required (aptt, drvvt, Kaolin clotting time, etc). 38 LABORATORY DIAGNOSIS OF ANTICARDIOLIPIN ANTIBODIES Harris and colleagues 9 were responsible for the development of an ELISA assay for the identification of ACA. A variety of commercially manufactured ELISA-based assays for ACA are available. Two types of microtiter plates are used: high-sensitivity plates (irradiated) and low-sensitivity plates. The high-sensitivity plates allow for greater antigenic density on the microtiter plate. Bivalent bonding of the antibodies in the patient plasma is critical to establish appropriate antigen-antibody density on the microtiter plate. Numerous commercial ACA ELISA systems are available. However, there is significant variability. The high-sensitivity microtiter plates that have been irradiated allow for greater antigenic density and facilitate bivalent bonding of antibodies present in patient plasma. In most cases, the antigenic target is 2 -GPI. However, there are significant antibodies to prothrombin in many patient plasmas. The most important variables in identifying a sensitive ACA system are concentration of target antigen (prothrombin, 2 -GPI, annexin V, etc), use of a high-sensitivity ELISA microtiter plate (irradiated), and an appropriate buffer system. Either serum or plasma may be used in testing for ACA. The term anticardiolipin antibody is a misnomer. As noted, the antibodies recognize proteins bound to the microtiter plate. In infectious disease states, one can see true ACAs (ie, bound to cardiolipin). Other approaches have been taken to identify ACAs, including flow cytometry. 42 Beads are coated with cardiolipin or specific proteins such as prothrombin or 2 -GPI. Using flow cytometry, one can identify all 3 isotypes (immunoglobulin [Ig] G, IgA, and IgM) in a single procedure. The drawbacks to the use of flow cytometry to identify ACA are requirements for significant laboratory volume of ACA testing, purchase cost of a flow cytometry system, and the current relative lack of commercially available flow cytometry kits to support ACA testing. Laboratories should carefully evaluate the various ELISA assays available for ACA testing. Based on interlaboratory external proficiency testing, the current state of the art in ACA testing is poor. There are a number of underlying variables that may explain the lack of concordance between laboratories. Among these variables are choice of microtiter plates for ELISA assays (high sensitivity vs lower sensitivity), technical issues within the laboratory, buffering reagents, and variable plate readers. CONCLUSIONS 1. Preanalytic variables, including preparation of PPP and freeze-thawing effects if plasma is frozen and tested at a later date, can influence the outcome of testing. 2. Establishing the diagnosis of LAs requires use of the guidelines as developed by the Scientific Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibodies (Table 5). 3. Integrated test systems are currently preferable when testing for LA. Examples include commercially available drvvt (screen)/drvvt (confirm; eg, increased phospholipid content). Other systems include Staclot LA, dilute prothrombin time/screen, and confirmatory reagent (dilute prothrombin time/high phospholipid). Venom-based assays include Textarin Time and Taipan Venom Time. 4. When performing ELISA assays, systems that use high-sensitivity microtiter plates are preferred. Alternatively, flow cytometry may offer a more sensitive and specific test system. RECOMMENDATIONS Lupus Anticoagulants/Antiphospholipid Antibodies Anticardiolipin antibody and LA assays are appropriate for patients with venous thromboembolism, particularly if the venous thromboembolism is idiopathic or associated with autoimmune disease, or if there is no family history of venous thrombosis. 29 Level 1 Anticardiolipin antibody and LA testing may be considered for patients with arterial thrombosis, particularly in a young person or a person with no documented atherosclerosis. 26,43 Level 1 Anticardiolipin antibody and LA assays should be considered for patients with unexplained stroke, particularly in a young person or a patient with autoimmune disease. 32,34 Level 1 Testing for LA and ACA should be considered for patients with cerebral venous thrombosis. 10 Level 3 Women with pregnancy loss that is either recurrent or late in the pregnancy (second and third trimester) should be evaluated for APAs (LA and APA). 31,44 46 Level 1 To demonstrate persistence, any positive test (APA or LA) must be confirmed by repeat testing after 6 weeks. 36 Level 2 Lupus Anticoagulants Platelet-poor plasma used for LA testing should have a platelet count less than 10 000/ L. 38 Level 1 The use of commercially available, integrated test systems for measuring LA is recommended, for example, the Staclot LA or drvvt. 41 Level 1 Patients being treated with anticoagulants and specimens containing anticoagulants should not be tested for LA; however, if patients on oral anticoagulants or heparin must be tested, the results must be interpreted with caution. 38 Level 2 Antiphospholipid Antibodies Immunoglobulin G ACA testing is recommended for the evaluation of thrombophilia. Although frequently measured, the risk of incident or recurrent thrombosis as- 1428 Arch Pathol Lab Med Vol 126, November 2002 Antiphospholipid Antibodies Triplett

sociated with IgM ACA and IgA ACA is uncertain. Elevated titers ( 40 GIU) are most closely associated with thrombophilia. 14 Level 2 Enzyme-linked immunosorbent assays for anti-prothrombin and anti- 2 GPI antibodies may be performed in addition to ACA testing in the evaluation of thrombophilia; however, prospective studies involving the use of assays for anti-prothrombin and anti- 2 GPI antibodies for the evaluation for thrombophilia are limited. 20 Level 3 For an explanation of the levels cited in this article, refer to Olson. 47 References 1. Wassermann A, Neisser A, Bruck C. Eine serodiagnostische Reaction bei Syphilis. Dtsch Med Wochensehr. 1906;32:745 746. 2. Pangborn MC. A new serologically active phospholipid from beef heart. Proc Soc Exp Biol Med. 1941;148:484 486. 3. Catterall RO. Biological false positive reactions and systemic disease. In: Walker G, ed. Ninth Symposium on Advanced Medicine. London, England: Pitman Medical; 1973:97 111. 4. Moore JE, Mohr CF. Biologically false positive serologic tests for syphilis: type, incidence, and cause. JAMA. 1952;150:467 473. 5. Conley CL, Hartmann RC. A hemorrhagic disorder caused by circulating anticoagulant in patients with disseminated lupus erythematosus. J Clin Invest. 1952:152:621 622. 6. Laurell AB, Nilsson IM. Hypergamma-globulinaemia, circulating anticoagulant, and biologic false positive Wassermann reaction: a study of two cases. J Lab Clin Med. 1957;49:694 707. 7. Feinstein DI, Rapaport SI. Acquired inhibitors of blood coagulation. Prog Hemost Thromb. 1972;1:75 95. 8. Jude B, Goudemand J, Dolle I, et al. Lupus anticoagulant: a clinical and laboratory study of 100 cases. Clin Lab Haematol. 1988;10:41 51. 9. Harris EN, Gharavi AE, Boey ML, et al. Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet. 1983;2:1211 1214. 10. Triplett DA, Brandt JT. Lupus anticoagulants: misnomer, paradox, riddle, epiphenomenon. Hematol Pathol. 1988;2:121 143. 11. Vila P, Hernandez MC, Lopez-Fernandez MT, Batlle J. Prevalence, followup and clinical significance of the anticardiolipin antibodies in normal subjects. Thromb Haemost. 1994:72:209 213. 12. Roubey RAS, Hoffman M. From antiphospholipid syndrome to antibody mediated thrombosis. Lancet. 1997;350:1491 1493. 13. Sugi T, McIntyre JA. Autoantibodies to phosphatidylethanolamine (PE) recognize a kininogen-pe complex. Blood. 1995;86:3083 3089. 14. Roubey RAS. Autoantibodies to phospholipid-binding plasma proteins: a new view of lupus anticoagulants and other antiphospholipid antibodies. Blood. 1994;84:2854 2867. 15. McNeil HP, Simpson RJ, Chesterman CN, Krilis SA. Anti-phospholipid antibodies are directed against a complex antigen that includes a lipid binding inhibitor of coagulation: 2 glycoprotein I (apolipoprotein H). Proc Natl Acad Sci USA.1990;87:4120 4124. 16. Warm H. Beta-2 glycoprotein I (apolipoprotein H) interactions with phospholipid vesicles. Int J Biochem. 1984;16:511 515. 17. Schousboe I. 2 : a plasma inhibitor of the contact activation of the intrinsic blood coagulation pathway. Blood. 1985;66:1086 1091. 18. Koike T, Ichikawa K, Kasahara H, Atsumi T, Tsutsumi A, Matsuura E. Epitopes on 2 -GPI recognized by anticardiolipin antibodies. Lupus. 1998;7(suppl 2):S14 S17. 19. McNeeley PA, Dlott JS, Furie RA, et al. 2 -glycoprotein I dependent anticardiolipin antibodies preferentially bind the amino terminal domain of 2 -glycoprotein I. Thromb Haemost. 2001;86:590 595. 20. degroot PG, Horbach DA, Simmelink MJA, Van Oort E, Derksen RHWM. Anti-prothrombin antibodies and their relation with thrombosis and lupus anticoagulants. Lupus. 1998;7:S32 S36. 21. Fleck RA, Rapport SI, Rao LVM. Antiprothrombin antibodies and the lupus anticoagulant. Blood. 1988;72:512 519. 22. Edson JR, Vogt JM, Hasegawa DK. Abnormal prothrombin cross-immunoelectrophoresis in patients with lupus inhibitors. Blood. 1984;64:807 816. 23. Corrigan JJ, Patterson JH, May ME. Incoagulability of the blood in systemic lupus erythematosus: a case due to hypoprothrombinemia and a circulating anticoagulant. Am J Dis Child. 1970;119:365 369. 24. Pratico D, Ferro D, Tuliano L, et al. Ongoing prothrombotic state in patients with antiphospholipid antibodies: a role for increased lipid peroxidation. Blood. 1999;93:3401 3407. 25. Shoenfeld Y, Viporen L. Lessons from experimental APS models. Lupus. 1998;7:S158 S161. 26. Finazzi G, Brancaccio V, Moia M, et al. Natural history and risk factors for thrombosis in 360 patients with antiphospholipid antibodies: a four year prospective study from the Italian registry. Am J Med. 1996;100:530 536. 27. Brey RL, Escalante A. Neurological manifestations of antiphospholipid antibody syndrome. Lupus. 1998;7:S67 S74. 28. Levine SR, Deegan MJ, Futrell N, Welch KMA. Cerebrovascular and neurologic disease associated with antiphospholipid antibodies: 48 cases. Neurology. 1990;40:1190 1196. 29. Ginsberg JS, Wells PS, Brill-Edwards P, et al. Antiphospholipid antibodies and venous thromboembolism. Blood. 1995;86:3685 3691. 30. Triplett DA, Asherson RA. Pathophysiology of the catastrophic antiphospholipid syndrome (CAPS). Am J Hematol. 2000;65:154 159. 31. Rand JH, Wu XX, Andree HA, et al. Pregnancy lost in the antiphospholipidantibody syndrome: a possible thrombogenic mechanism. N Engl J Med. 1997; 337:154 160. 32. Levine SR, Salowich-Palm L, Sawaya KL, et al. IgG anticardiolipin antibody titer 40 GPL and the risk of subsequent thrombo-occlusive events and death: a prospective cohort study. Stroke. 1997;28:1660 1665. 33. Golden AL. Livedo reticularis in systemic lupus erythematosus. Arch Dermatol. 1963;87:299 301. 34. The Antiphospholipid Antibodies in Stroke Study Group (APSAA). Anticardiolipin antibodies are an independent risk factor for first ischemic stroke. Neurology. 1993;43:2069 2073. 35. Harris EN, Bos K. An acute disseminated coagulopathy-vasculopathy associated with the antiphospholipid syndrome. Arch Intern Med. 1991;151:231 233. 36. Hughes GRV, Harris EN, Gharavi AE. The anticardiolipin syndrome. J Rheumatol. 1986;13:486 489. 37. Triplett DA, Brandt JT, Kaczor D, Schaeffer J. Laboratory diagnosis of lupus inhibitors: a comparison of the tissue thromboplastin inhibition procedure with a new platelet neutralization procedure. Am J Clin Pathol. 1983;79:678 682. 38. Brandt JT, Triplett DA, Alving B, Scharrer I. Criteria for the diagnosis of lupus anticoagulants: update. Thromb Haemost. 1995;74:1185 1190. 39. Thiagarajan P, Pengo V, Shapiro SS. The use of the dilute Russell viper venom time for the diagnosis of lupus anticoagulants. Blood. 1986;68:869 874. 40. Lawrie AS, Mackie IJ, Purdy G, Machin SJ. The sensitivity and specificity of commercial reagents for the detection of lupus anticoagulants show marked differences in performance between photo-optical and mechanical coagulometers. Thromb Haemost. 1999;81:758 762. 41. Triplett DA, Barna LK, Unger GA. A hexagonal (II) phase phospholipid neutralization assay for lupus anticoagulant identification. Thromb Haemost. 1993;70:787 793. 42. Stewart MW, Etches WS, Russell AS, et al. Detection of antiphospholipid antibodies by flow cytometry, rapid detection of antibody isotype and phospholipid specificity. Thromb Haemost. 1993;70:603 607. 43. Van Cott EM, Laposata M, Prins MH. Laboratory evaluation of hypercoagulability with venous or arterial thrombosis: venous thromboembolism, myocardial infarction, stroke, and other conditions. Arch Pathol Lab Med. 2002;126: 1281 1295. 44. Vinatier D, Dufour P, Cosson M, et al. Antiphospholipid syndrome and recurrent miscarriages. Eur J Obstet Gynecol Reprod Biol. 2001;96:37 50. 45. Yamada H, Kato EH, Ebina Y, et al. Recurrent pregnancy loss: etiology of thrombophilia. Semin Thromb Hemost. 2001;27:121 129. 46. Lee RM, Branch DW, Silver RM. Immunoglobulin A anti-beta-2-glycoprotein antibodies in women who experience unexplained recurrent spontaneous abortion and unexplained fetal death. Am J Obstet Gynecol. 2001;185:748 753. 47. Olson JD. College of American Pathologists Consensus Conference XXXVI: Diagnostic Issues in Thrombophilia: introduction and general considerations. Arch Pathol Lab Med. 2002;126:1277 1280. Arch Pathol Lab Med Vol 126, November 2002 Antiphospholipid Antibodies Triplett 1429