ORIGINAL ARTICLE. Abstract. Introduction

Transcription

1 ORIGINAL ARTICLE Presence of Antiphospholipid Antibodies as a Risk Factor for Thrombotic Events in Patients with Connective Tissue Diseases and Idiopathic Thrombocytopenic Purpura Koji Habe 1,HideoWada 2, Takeshi Matsumoto 3, Kohshi Ohishi 3, Makoto Ikejiri 4, Kimiko Matsubara 1, Tatsuhiko Morioka 1, Yuki Kamimoto 5, Tomoaki Ikeda 5, Naoyuki Katayama 6 and Hitoshi Mizutani 1 Abstract Objective Antiphospholipid syndrome (APS) is a well-known complication of habitual abortion and/or thrombosis and is frequently associated with autoimmune diseases. Methods We retrospectively investigated the relationships between the presence of antiphospholipid antibodies (apls) and the incidence of thrombotic events (THEs) in 147 patients with various connective tissue diseases (CTD) suspected of having APS and 86 patients with idiopathic thrombocytopenic purpura (ITP). THEs were observed in 41 patients, including 14 cases of venous thrombosis, 21 cases of arterial thrombosis and eight cases of complications of pregnancy. Results The prevalence of THE was significantly high in the systemic lupus erythematosus (SLE) patients compared with the other CTD patients and ITP patients. The frequency of lupus anticoagulant (LA), anticardiolipin antibodies (acl)-β2-glycoprotein (GPI) complex IgG and apl was significantly high in the SLE patients compared with the ITP patients. Subsequently, the rate of development of THE was significantly high in the patients with apls. In particular, the incidence of THE was significantly high in the SLE or ITP patients with LA, acl-β2gpi IgG or apl. The optimal cut-off values for LA, acl IgG and acl-β2gpi complex IgG for the risk of THEs were higher in the SLE patients in comparison to the values obtained when using the kit provided by the manufacturer. Conclusion Although apls is frequently associated with SLE and is a causative factor for thrombosis, the optimal cut-off value for apl for predicting the occurrence of THEs varies among different underlying diseases. Key words: APS, thrombosis, SLE, LA, β2-glycoprotein I (Intern Med 55: , 2016) () Introduction Antiphospholipid syndrome (APS) (1, 2) is a well-known systemic thrombotic diathesis associated with the presence of antiphospholipid antibodies (apls). The mechanisms underlying the development of thrombosis, including cerebral thrombosis ( CT ) (3) and venous thromboembolism (VTE) (4), in addition to obstetric morbidity (5) due to apls are poorly understood. apls derived from patients with APS preferentially target negatively charged phospholipids (PLs) and/or their complex formed with plasma proteins, including Department of Dermatology, Mie University Graduate School of Medicine, Japan, Department of Molecular and Laboratory Medicine, Mie University Graduate School of Medicine, Japan, Blood Transfusion Service, Mie University Graduate School of Medicine, Japan, Central Laboratory, Mie University Hospital, Japan, Department of Obstetrics and Gynecology, Mie University Graduate School of Medicine, Japan and Department of Hematology and Oncology, Mie University Graduate School of Medicine, Japan Received for publication April 6, 2015; Accepted for publication June 2, 2015 Correspondence to Dr. Hideo Wada, wadahide@clin.medic.mie-u.ac.jp 589

2 Table 1. The Thrombotic Events. CTD ITP Cerebral thrombosis 5 3 Cerebral thrombosis, skin ulcer 1 0 Infarction of spinal cord 1 0 Transient ischemic attack 1 0 Skin ulcer 6 0 Pulmonary embolism, DVT 6 3 Budd - Chiari syndrome, DVT 1 0 Cerebral thrombosis, DVT 2 0 Pulmonary embolism, skin ulcer 1 0 Thrombosis of the artery and veins of the retina 1 0 Thrombosis of shunt 1 0 Disseminated intravascular coagulation 1 0 Unexplained abortions beyond Pregnancy 10 th weeks gestation 5 0 complication Unexplained premature birth before the 34 th week of gestation DVT: deep vein thrombosis, CTD: connective tissue diseases β2-glycoprotein I (β2gpi) (6). Clinical laboratory tests for apls include those for anticardiolipin antibodies (acl), lupus anticoagulant (LA) and anti-β2gpi antibodies (7). These antibodies have different patterns of sensitivity and specificity with respect to the incidence of thrombotic events (THEs). APS consists of primary APS and secondary APS (8, 9). The underlying diseases of secondary APS include connective tissue diseases (CTD), such as systemic lupus erythematosus (SLE) (10), as well as related autoimmune diseases, such as idiopathic thrombocytopenic purpura (ITP) (11). The frequency of apls is reported to be 20-50% in patients with SLE (12-14), which is slightly higher than that seen in those with systemic scleroderma (SSc) (15, 16), Sjögren s syndrome (SjS) (17) and/or rheumatoid arthritis (RA) (18, 19). The relationship between apls and THEs has been previously reported, suggesting that the risk of THE increases as the number of positive apls increases (20-22). The current study retrospectively investigated the relationship between the presence of apls and the incidence THEs in 147 patients with CTD and 86 patients with ITP who were clinically suspected of having APS. Materials and Methods Laboratory data were continuously examined for 147 patients with CTD and 86 patients with ITP who consulted the Department of Hematology or Department of Dermatology between January 1, 1994 and December 31, SLE (23), SSc (24) and Sjogren s syndrome (25) were diagnosed according to the diagnostic criteria of the American College of Rheumatology. ITP was diagnosed based on the criteria established by the American Society of Hematology (26). Disseminated intravascular coagulation (DIC) was diagnosed based on the overt-dic diagnostic criteria established by the International Society of Thrombosis and Haemostasis (ISTH) (27). VTE was diagnosed using echography, venography or computed tomography (CT). Cerebral vascular disease was diagnosed using CT or magnetic resonance imaging (MRI), and cerebral venous sinus thrombosis (CVST) was diagnosed based on MRI, magnetic resonance venography (MRV) or cerebral angiography (CAG). Vasculitis was diagnosed with CT and MRI. The subjects with CTD (with and without THE) included 54 patients with SLE (22 and 32), 47 patients with SSc (5 and 42), 15 patients with overlap syndrome (3 and 12), 15 patients with SjS (5 and 10), 11 patients with dermatomyositis (0 and 11), five patients with mixed connective tissue disease (MCTD; 0 and 5). The patients with ITP included six patients with THEs and 80 patients without THEs. The study protocol was approved by the Human Ethics Review Committee of Mie University School of Medicine (No. 2464), and informed consent was obtained. The laboratory data obtained at the time that collagen disease was suspected were examined in this study. The lupus anticoagulant (LA) titer was measured using diluted Russell s viper venom time based on the LA test Gradipore (Medical and Biological Laboratories CO., LTD.; MBL, Nagoya, Japan). The titers of anticardiolipin IgG antibodies (acl-igg) and anticardiolipin β2-gpi complex IgG antibodies (acl-β2-gpi complex IgG) were measured according to an enzyme-linked immunosorbent assay (ELISA) using a MESACUP cardiolipin IgG test (MBL) and anti-cl β2gpi kit Yamasa EIA (Yamasa Shoyu, Tyoushi, Japan) (28). Statistical analysis The data are expressed as the medians (5th-95th percentile). Differences between the groups were examined for statistical significance using the Mann-Whitney U-test. A p value of <0.05 denoted the presence of a statistically significant difference. In addition, the significance of the frequency rates was examined using a Chi-square analysis and Fisher s exact test. Results THEs were observed in 35 patients with CTD and six patients with ITP, including 14 cases of VTE, 21 cases of arterial thrombosis and eight pregnancy complications (Table 1). The cases of VTE included 12 patients with deep vein thrombosis (DVT), 10 patients with PE and one patient with thrombosis of the vein of the retina. The cases of arterial thrombosis included 11 patients with cerebral thrombosis and seven patients with skin ulcers due to skin arterial thrombosis. The frequency of THEs was significantly higher in the patients with CTD, especially those with SLE, compared to those with ITP; however, there were no significant differences in the frequency of THEs between the patients with CTD without SLE and those with ITP. The frequency of LA (p<0.01), acl-β2gpi complex IgG (p<0.001) and apl (p<0.001) was significantly higher in the patients with SLE than in those with ITP (Table 2). Only the rate of apl 590

3 Table 2. The Presence of LA, acl IgG, acl-β2-gpi Complex IgG and apl in CTD Patients, SLE Patients, CTD Patients without SLE, and ITP Patients. CTD ITP Odds ratio positive / negative LA 18 / 90 6 / ( ) p = 0.25 acl IgG 18 / / ( ) p = 0.88 acl- -GPI complex IgG 25 / / ( ) p = 0.06 apl 44 / / ( ) p < 0.01 SLE ITP LA 16 / 30 6 / ( ) p < 0.01 acl IgG 10 / 23 2 / ( ) p = 0.70 acl- -GPI complex IgG 17 / 32 6 / ( ) p < apl 28 / / ( ) p < CTD without SLE ITP LA 2 / 60 6 / ( ) p = 0.30 acl IgG 8 / 80 2 / ( ) p = 0.68 acl- -GPI complex IgG 8 / 80 6 / ( ) p = 0.97 apl 16 / / ( ) p = 0.40 CTD: Connective tissue disease, SLE: systemic erythematosus, ITP: idiopathic thrombocytopenic purpura, LA: lupus anticoagulant, acl: anticardiolipin antibodies, acl- -GPI complex IgG: acl- -glycoprotein I complex IgG antibody, apl: antiphospholipid antibodies Table 3. The Odds Ratios for the Risk of Thrombosis Due to the Presence of Antiphospholipid Antibodies in CTD Patients, SLE Patients, CTD Patients without SLE and ITP Patients. CTD SLE CTD without SLE With THE Without THE Odds ratio positive / negative LA 14 / 16 4 / ( ), p < acl IgG 10 / 16 8 / ( ), p < acl- -GPI complex IgG 16 / 16 9 / ( ), p < apl 20 / / ( ), p < 0.01 LA 12 / 7 4 / ( ), p < 0.01 acl IgG 6 / 8 4 / ( ), p = 0.33 acl- -GPI complex IgG 13 / 8 4 / ( ), p < 0.01 apl 18 / 4 10 / ( ), p < LA 2 / 8 0 / 52 - acl IgG 4 / 8 4 / ( ), p < 0.01 acl- -GPI complex IgG 3 / 8 5 / ( ), p = 0.09 apl 7 / 6 9 / ( ), p < LA 3 / 3 3 / ( ), p < 0.05 acl IgG 0 / 0 2 / 9 - ITP acl- -GPI complex IgG 3 / 3 3 / ( ), p < 0.01 apl 4 / 2 6 / ( ), p < CTD: Connective tissue disease, SLE: systemic lupus erythematosus, ITP: idiopathic thrombocytopenic purpura, LA: lupus anticoagulant, acl: anticardiolipin antibodies, acl- -GPI complex IgG: acl- -glycoprotein I complex IgG antibodies, apl: antiphospholipid antibodies was significantly higher in the patients with CTD than in those with ITP (p<0.01). There were no significant differences in the frequence of LA, acl IgG, acl-β2gpi complex IgG and apl between the CTD patients without SLE and the ITP patients. The presence of LA, acl IgG, aclβ2gpi complex IgG and apl was each associated with a higher risk of THEs in the overall CTD patients. In addition, the presence of LA, acl-β2-gpi complex IgG and apl was associated with a higher risk of THEs in SLE patients, while the presence of LA, acl-β2gpi complex IgG and apl was associated with a higher risk in the ITP patients (Table 3). The LA, acl IgG and acl-β2gpi complex IgG levels were significantly higher in the patients with THEs than in those without THEs (Fig. 1). In the receiver operating characteristics (ROC) analysis, the cut-off value of LA for the risk of THEs (all patients: 1.72) was high in the SLE patients (1.60) and similar in the ITP patients (1.28) in comparison to that determined by the kit provided by the manufacturer (1.30). The optimal cut-off value of acl IgG for the risk of THEs (all patients: 16.0 U/mL) was high in 591

4 a b Table 4. The Cut-off Values for the Risk of Thrombosis. LA acl IgG ( U / ml ) acl- 2-GPI complex IgG ( U / ml ) Cut-off value AUC Odds ratio CTD SLE CTD without SLE ITP All patients CTD SLE CTD without SLE ITP All patients CTD SLE CTD without SLE ITP All patients Cut-off value provided by the manufacturer ARC: area under the curve, CTD: Connective tissue disease, SLE: systemic lupus erythematosus, ITP: idiopathic thrombocytopenic purpura (Fig. 2). Discussion c Figure 1. LA, acl IgG and acl-β2-gpi complex IgG levels in the patients with CTD, SLE and ITP. a: LA, b: acl IgG, c: acl-β2-gpi complex IgG, THE: thrombotic events. ***: p<0.001, **: p<0.01, *: p<0.05 the SLE cases (20 U/mL) and similar in the CTD patients without SLE (12 U/mL) in comparison to that determined by the kit provided by the manufacturer (10 U/mL). The cut-off value of the acl-β2gpi complex IgG for the risk of THEs (all patients: 6.8 U/mL) was high in the CTD (8.67 U/mL), SLE (10.29 U/mL), CTD without SLE (8.62) and ITP (6.47 U/mL) cases in comparison to that determined by the kit provided by the manufacturer (3.5 U/mL)(Table 4). The correlations between LA and acl IgG (r= 0.536, p< 0.001) and between LA and acl-β2-gpi complex IgG (r= 0.527, p<0.001) were similar (neither of the pairs were well correlated). The level of acl IgG was well correlated with the level of acl-β2-gpi complex IgG (r= 0.785, p<0.001) The presence of apls has been reported to be associated with a high risk of thrombosis (1, 2, 22, 29). Our previous study (22) reported that the rate of VTE is significantly higher than that of cerebral thrombosis in patients with suspected APS. However, that study included many primary APS patients with a limited number of SLE patients. The present study focused on THE in CTD patients. Consequently the rate of arterial thrombosis was highest in the SLE patients among the patients with CTD. A predominance of arterial thrombosis has been reported in Japanese patients with primary APS, a study that included 50.4% of SLE patients (30). Therefore, the presence of apls in patients with CTD, particularly SLE, indicates a high risk of arterial thrombosis, such as cerebral thrombosis. Even not as high as that seen in cases of SLE, patients with other autoimmune diseases with apls also carry a high risk of VTE. In this study, six of the 86 patients with ITP had associated thrombosis. A few previous cases of ITP have also been reported to be associated with thrombosis (31). These findings suggest that some cases of ITP associated with APS, and/or some cases of ITP, may transform to SLE. In this study, the frequency of LA or acl-β2-gpi IgG was significantly higher in the patients with SLE, but not in the overall patients with CTD, compared with that observed in the ITP patients, suggesting a strong association between SLE and apl. However, no differences in the incidence of acl IgG were observed among the SLE, CTD without SLE and ITP groups. The presence of apls has been reported in approximately 40% of SLE patients, and half of these patients experience THEs or obstetric morbidities (32). The frequency of apl is reported to be 0-4 % in healthy people (33, 34), and this rate is slightly higher in the patients with ITP (35). The analysis of correlations among the apls 592

5 a b c Figure 2. Correlations among LA, acl IgG and acl-β2- GPI complex IgG. a: The relationship between LA and acl IgG (Y= X). b: The relationship between LA and acl-β2-gpi complex IgG (Y= X). c: The relationship between acl IgG and acl-β2-gpi complex IgG (Y= X). groups in this study suggested that LA may be independent of other apl parameters and that acl-β2gpi IgG may overlap with acl IgG. In the present analysis, the frequency of THE was significantly high among the patients positive for LA, acl IgG, acl-β2gpi IgG or apl in the CTD group, the patients positive for LA, acl-β2gpi IgG or apl in the SLE and ITP groups and the patients positive for acl IgG or apl in the CTD group without SLE. These findings suggest that LA is the most useful apl for predicting THEs in CTD patients and that acl-β2-gpi complex IgG may be the most useful apl for predicting THEs in ITP patients. A systemic review indicated that LA is associated with a higher risk of THE than acl IgG (36). Furthermore, our previous study of APS patients showed that the incidence of THEs is related to the presence of LA, but not the acl-β2-gpi complex IgG or acl-igg (22). The LA test includes different measurement parameters, including dilute Russell viper venom test (DRVVT) and partial thromboplastin time (PTT)-LA (37). Therefore, the results may differ depending the assay system used (38). The presence of LA reflects abnormalities in various apls, except for anti-β2gpi antibodies and antiprothrombin antibodies (39). An international multicenter study reported that the results of antiβ2gpi-dependent lupus anticoagulant (LAC) assays correlate with the presence of thrombosis better than the findings of the classic LAC assay, such as that for acl IgG (40, 41). On the contrary, the acl IgG titer is reported to have a higher predictive value for thrombosis than the anti-β2gpi antibody titer (41). The predictive value of the presence of apls for thrombosis in CTD may differ among underlying disease. Cases with dual or triple positive results for LA, anti-β2gpi antibodies and acl are strongly associated with the development of thrombosis and pregnancy morbidities (42, 43). In the present study, the optimal cut-off values of LA, acl IgG and acl-β2-gpi complex IgG for the risk of THEs were higher in the SLE cases, but similar in the ITP (1.28) cases, in comparison to that determined by the kit provided by the manufacturer (1.30), suggesting that the optimal cut-off value of apls for predicting VTEs should be determined based on the underlying disease. Although our study showed the usefulness of each apl for obtaining a diagnosis of THEs, the combination of several apls might also be useful. The efficacy of the antiphospholipid score for diagnosing APS and predicting THEs has been reported (44) and the apl scoring system has thus been suggested to be useful. There are many risk factors for thrombosis other than apls, such as diabetes, atherosclerotic factors, genetic factors, cigarette smoking and aging in patients with CTD. Obesity is also a risk factor for VTE and acute myocardial infarction (45). Multivariate studies including assessments of atherosclerotic factors will be useful for investigating the risk of thrombosis in these patients. In conclusion, the presence of apls is closely associated with the development of THEs in CTD. In particular, LA is a very strong risk factor for THE in SLE patients. Moreover,thepresenceofaCL-β2-GPI complex IgG is a potent risk factor for THE in ITP patients, and the optimal cut-off values for predicting THEs are different among patients with different underlying diseases. The authors state that they have no Conflict of Interest (COI). 593

6 Acknowledgement Our work is supported in part by research grants from the Japanese Ministry of Health, Labour and Welfare and the Japanese Ministry of Education, Science, Sports and Culture. References 1. Palomo IG, Segovia FM, Alarcon ML, et al. An insight into the pathophysiology of thrombosis in antiphospholipid syndrome. Front Biosci 12: , Staub HL, Bertolaccini ML, Munther MA. Antiphosphatidylethanolamine antibody, thromboembolic events and the antiphospholipid syndrome. Autoimmun Rev 52: , Muscal E, Brey RL. Antiphospholipid syndrome and the brain in pediatric and adult patients. Lupus 19: , Hughes G. Hughes Syndrome: the antiphospholipid syndrome-a clinical overview. Clin Rev Allergy Immunol 32: 3-12, Chandramouli NB, Rodgers GM. Management of thrombosis in women with antiphospholipid syndrome. Clin Obstet Gynecol 44: 36-47, Ruiz-Irastorza G, Crowther M, Branch W, Khamashta MA. Antiphospholipid syndrome. Lancet 376: , Ortel TL. Antiphospholipid syndrome: laboratory testing and diagnostic strategies. Am J Hematol 87: S75-S81, Asherson RA, Khamashta MA, Ordi-Ros J, et al. The primary antiphospholipid syndrome; major clinical and serological features. Medicine 68: , Alarcon-Segovia D, Deleze M, Oria CV, Sanchez-Guerrero J, Gomez-Pacheco L. Antiphospholipid antibodies and the antiphospholipid syndrome in SLE. A prospective analysis of 500 consecutive patients. Medicine 68: , Avcin T, Silverman ED. Antiphospholipid antibodies in pediatric systemic lupus erythematosus and the antiphospholipid syndrome. Lupus 16: , Atsumi T, Furukawa S, Amengual O, Koike T. Antiphospholipid antibody associated thrombocytopenia and the paradoxical risk of thrombosis. Lupus 14: , Tubach F, Hayem G, Marchand JL, et al. IgG anti-beta2- glycoprotein I antibodies in adult patients with systemic lupus erythematosus: prevalence and diagnostic value for the antiphospholipid syndrome. J Rheumatol 27: , Long AA, Ginsberg JS, Brill-Edwards P, et al. The relationship of antiphospholipid antibodies to thromboembolic disease in systemic lupus erythematosus: A cross-sectional study. Thromb Haemost 66: , Sebastiani GD, Passiu G, Galeazzi M, Porzio F, Carcassi U. Prevalence and clinical associations of anticardiolipin antibodies in systemic lupus erythematosus: a prospective study. Clin Rheumatol 10: , Gupta R, Thabah MM, Gupta S, Shankar S, Kumar A. Clinical significance of antiphospholipid antibodies in Indian scleroderma patients. Rheumatol Int 30: , Marie I, Jouen F, Hellot MF, Levesque H. Anticardiolipin and anti-beta2 glycoprotein I antibodies and lupus-like anticoagulant: prevalence and significance in systemic sclerosis. Br J Dermatol 158: , Pasoto SG, Chakkour HP, Natalino RR, et al. Lupus anticoagulant: a marker for stroke and venous thrombosis in primary Sjögren s syndrome. Clin Rheumatol 31: , Unikiene D, Misiūniene N. Antiphospholipid syndrome in patients with systemic connective tissue diseases. Medicina (Kaunas) 39: , Olech E, Merrill JT. The prevalence and clinical significance of antiphospholipid antibodies in rheumatoid arthritis. Curr Rheumatol Rep 8: , Neville C, Rauch J, Kassis J, et al. Thromboembolic risk in patients with high titre anticardiolipin and multiple antiphospholipid antibodies. Thromb Haemost 90: , Pengo V, Ruffatti A, Legnani C, et al. Incidence of a first thromboembolic event in asymptomatic carriers of high-risk antiphospholipid antibody profile:a multicenter prospective study. Blood 118: , Habe K, Wada H, Matsumoto T, et al. Presence of antiphospholipid antibody is a risk factor in thrombotic events in patients with antiphospholipid syndrome or relevant diseases. Int J Hematol 97: , Smith EL, Shmerling RH. The American College of Rheumatology criteria for the classification of systemic lupus erythematosus: strengths, weaknesses, and opportunities for improvement. Lupus 8: , Subcommittee for Scleroderma Criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. Preliminary criteria for the classification of systemic sclerosis (scleroderma). Arthritis Rheum 23: , Rasmussen A, Ice JA, Li H, et al. Comparison of the American- European Consensus Group Sjögren s syndrome classification criteria to newly proposed American College of Rheumatology criteria in a large, carefully characterised sicca cohort. Ann Rheum Dis 73: 31-38, George JN, Woolf SH, Raskob GE, et al. Idiopathic thrombocytopenic purpura: a practice guide devel oped by explicit methods for the American Society of Hematology. Blood 88: 3-40, Taylor FB Jr, Toh CH, Hoots WK, Wada H, Levi M. Towards definition, clinical and laboratory criteria, and a scoring system for disseminated intravascular coagulation. On behalf of the Scientific Subcommittee on disseminated intravascular coagulation (DIC) of the International Society on Thrombosis and Haemostasis (ISTH). Thromb Haemost 86: , Kaburaki J, Kuwana M, Ikeda Y. Anti-cardiolipin-beta2-GPI complex antibodies in idiopathic thrombocytopenic purpura. Intern Med 37: 796, Muñoz-Rodriguez FJ, Font J, Cervera R, et al. Clinical study and follow-up of 100 patients with the antiphospholipid syndrome. Semin Arthritis Rheum 29: , Fujieda Y, Atsumi T, Amengual O, et al. Predominant prevalence of arterial thrombosis in Japanese patients with antiphospholipid syndrome. Lupus 21: , Zhou H, Fu R, Wang H, et al. Immune thrombocytopenia in the elderly: clinical course in 525 patients from a single center in China. Ann Hematol 92: 79-87, Galli M, Luciani D, Bertolini G, Barbui T. Lupus anticoagulants are stronger risk factors for thrombosis than anticardiolipin antibodies in the antiphospholipid syndrome:a systemtic review of the literature. Blood 101: , Ghosh K, Shetty S, Mohanty D. Detection of unexpected lupus anticoagulant during preparation of normal pooled plasma (NPP) from healthy volunteers. Thromb Haemost 84: , Gris JC, Perneger TV, Quéré I, et al. Antiphospholipid/antiprotein antibodies, hemostasis-related autoantibodies, and plasma homocysteine as risk factors for a first early pregnancy loss: a matched case-control study. Blood 102: , Stasi R, Stipa E, Masi M, et al. Prevalence and clinical significance of elevated antiphospholipid antibodies in patients with idiopathic thrombocytopenic purpura. Blood 84: , Jennings I, Kitchen S, Kitchen DP, Woods TA, Walker ID. ISTH/ SSC lupus anticoagulant testing guidelines: how far have hese been adopted by laboratories? J Thromb Haemost 9: , Kandiah DA, Krilis SA. Heterogeneity of lupus anticoagulant (LA) antibodies: LA activity in dilute Russell s viper venom time and dilute kaolin clotting time detect different populations of anti- 594

7 bodies in patients with the antiphospholipid syndrome. Thromb Haemost 80: , Pengo V, Denas G, Bison E, et al; Italian Federation of Thrombosis Centers (FCSA). Prevalence and significance of antiprothrombin (apt) antibodies in patients with lupus anticoagulant (LA). Thromb Res 126: , De Laat B, Derksen RH, Reber G, et al. An international multicentre-laboratory evaluation of a new assay to detect specifically lupus anticoagulants dependent on the presence of anti-beta 2-glycoprotein autoantibodies. J Thromb Haemost 9: , Pengo V. APS controversies in diagnosis and management, critical overview of current guidelines. Thromb Res 127: S51-S52, Deng X, Liu X. Reevaluation of predictive value of ACL and anti- 2GP1 antibody for thrombosis in patients with systemic lupus erythematosus: from a perspective of a practical world. Rheumatol Int 32: , Sciascia S, Murru V, Sanna G, Roccatello D, Khamashta MA, Laura Bertolaccini M. Clinical accuracy for diagnosis of Antiphospholipid Syndrome in SLE: evaluation of 23 possible combinations of antiphospholipid antibody specificities. J Thromb Haemost 10: , Nojima J. Advanced clinical laboratory studies in the graduate school of medicine-studies on pathogenic mechanisms of antiphospholipid syndrome. Rinsho Byori (The Japanese Journal of Clinical Pathology) 57: , 2009 (in Japanese, Abstract in English). 44. Otomo K, Atsumi T, Amengual O, et al. Efficacy of the antiphospholipid score for the diagnosis of antiphospholipid syndrome and its predictive value for thrombotic events. Arthritis Rheum 64: , Horvei LD, Brækkan SK, Mathiesen EB, Njølstad I, Wilsgaard T, Hansen JB. Obesity measures and risk of venous thromboembolism and myocardial infarction. Eur J Epidemiol 29: , The Japanese Society of Internal Medicine 595