nti-phospholipid antibodies (apl) are a heterogeneous

Transcription

1 Prevalence and Clinical Correlation of Anti-Phospholipid Binding Protein Antibodies in Anticardiolipin-Negative Patients With Systemic Lupus Erythematosus and Women With Unexplained Recurrent Miscarriages Nicola Bizzaro, MD; Elio Tonutti, MD; Danilo Villalta, MD; Marilina Tampoia, MD; Renato Tozzoli, MD Context. Anti-phospholipid antibodies (apl) are a heterogeneous group of autoantibodies, the presence of which is associated with thrombotic events and miscarriage. Objective. To establish whether antibodies directed against phospholipid-binding plasma proteins such as 2 - glycoprotein I ( 2 GPI), prothrombin (PT), and annexin V (Anx V) constitute a risk factor for thromboembolism in patients with systemic lupus erythematosus (SLE) and for miscarriage in women with recurrent pregnancy loss (RPL), independently of the presence of the classic anticardiolipin (acl) antibodies, and whether their determination together with that of acl would help to increase the diagnostic sensitivity of apl tests. Design. The prevalence of various antibodies directed toward phospholipids (CL and other anionic phospholipids [APL]) and phospholipid-binding proteins ( 2 GPI, PT, and Anx V) was determined by immunoenzymatic methods in 311 serum samples. Patients. Twenty-five patients with acl-positive primary anti-phospholipid syndrome (paps); 89 patients with SLE, 23 of whom had thrombotic complications (SLE/APS) and 66 of whom had no thrombosis; and 77 women with unexplained recurrent pregnancy loss comprised our study group. One hundred twenty healthy subjects matched for age and sex were studied as the control group. Results. Immunoglobulin (Ig) G and/or IgM aapl, anti- 2 GPI, anti-pt, and IgG anti-anx V antibodies were detected in 25 (100%), 20 (80%), 15 (60%), and 6 (24%), respectively, of the 25 acl-positive paps patients; IgG and/or IgM acl, aapl, anti- 2 GPI, anti-pt, and IgG anti- Anx V antibodies were detected in 33 (37%), 42 (47%), 31 (35%), 40 (45%), and 12 (13%) of the 89 SLE patients, respectively. Of the 56 SLE patients who proved to be acl negative, anti- 2 GPI was present in 3 patients (5%), anti- PT in 13 (23%) patients, and anti-anx V in 5 (9%) patients. In the subset of 23 SLE/APS patients, IgG anti-pt prevalence was higher than that of the other autoantibodies (87% vs 70% acl, 66% aapl, 57% anti- 2 GPI, and 4% anti-anx V), and in 26% of cases, IgG anti-pt was the only antibody present. Anti-PT had a slightly lower specificity than acl (46% vs 49%); however, the occurrence of both antibodies brought the specificity to 92.4%. The highest risk for thrombosis in SLE patients was associated with the presence of IgG anti-pt antibody (odds ratio [OR] 15.3, P.001, vs 6.5 acl, 3.5 aapl, 3.4 anti- 2 GPI, 0.2 anti-anx V). Fifty-one of the 77 women with recurrent pregnancy loss were negative for all antibodies investigated; the prevalence of IgG and/or IgM acl, aapl, anti- 2 GPI, anti-pt, and IgG anti-anx V antibodies was 6% (5), 12% (9), 6% (5), 16% (12), and 17% (13), respectively. Of the 67 aclnegative women, none had anti- 2 GPI antibodies, 7 (11%) were anti-pt positive, and 13 (19%) were anti-anx V positive. In the subgroup of 26 recurrent pregnancy loss patients who had at least one antibody, anti-anx V was present in 50% of cases (in 42% as the sole antibody) and was the only antibody significantly associated with miscarriage (P.02). Conclusions. The results of this study indicate that it is useful to measure anti-pt antibodies in addition to the more widely used acl and anti- 2 GPI antibodies in the prognostic evaluation of SLE patients for the risk of thrombosis, and the results also confirm that anti-anx V antibodies may play an important role in recurrent pregnancy loss. (Arch Pathol Lab Med. 2005;129:61 68) Accepted for publication August 6, From the Laboratorio di Patologia Clinica, Ospedale di S. Donà di Piave, Italy (Dr Bizzaro); Immunologia e Allergologia, Az. Osp. S. Maria della Misericordia, Udine, Italy (Dr Tonutti); Servizio di Immunologia Clinica e Virologia, Az. Osp. S. Maria degli Angeli, Pordenone, Italy (Dr Villalta); Laboratorio di Patologia Clinica, Policlinico, Bari, Italy (Dr Tampoia); and the Laboratorio di Chimica-clinica e Microbiologia, Ospedale di Latisana, Italy (Dr Tozzoli). The authors have no relevant financial interest in the products or companies described in this article. Reprints: Nicola Bizzaro, MD, Laboratorio di Patologia Clinica, Ospedale Civile, Via N. Sauro, 25, S.Donà di Piave (VE), Italy ( nbizzaro@dacos.it). A nti-phospholipid antibodies (apl) are a heterogeneous group of autoantibodies, the persistent presence at medium-high levels of which is associated with the occurrence of thromboembolic events and fetal loss. Those apl most commonly used in clinical practice are utilized in tests for anticardiolipin antibodies (acl) and lupus anticoagulant (LA), which constitute one of the criteria for classification of primary anti-phospholipid syndrome (paps) 1 and systemic lupus erythematosus (SLE). 2 However, the demonstration that one of the main target antigens of apl actually consists of 2 -glycoprotein I ( 2 GPI), a plasma protein involved in coagulation processes, 3,4 has shifted researchers attention to the study of other anti- Arch Pathol Lab Med Vol 129, January 2005 Anti-Phospholipid Binding Protein Antibodies Bizzaro et al 61

2 bodies directed toward coagulation proteins, some of which, like prothrombin (PT) and annexin V (Anx V), act as phospholipid cofactors. It is now generally believed that it is not phospholipids, but rather phospholipid-binding proteins or phospholipid-protein complexes, that constitute the real target of apl. The action mechanism of anti-phospholipid cofactor antibodies is not yet fully understood, but they probably react with neoepitopes, which form as a result of the protein-phospholipid bond. The antibodies thus seem able to interfere with the pro- or anticoagulant reactions that occur in the cell membranes 5,6 and the vascular endothelium cells 7,8 and to be responsible for the high incidence of thrombotic events in APS patients. Anti- 2 GPI antibodies are found in approximately 30% of SLE patients and constitute an independent risk factor for arterial or venous thrombosis Anti-prothrombin antibodies are found in a higher percentage (50% 60%) of SLE patients than are acl and anti- 2 GPI antibodies, 12 and they also constitute an independent risk factor for venous thrombosis The simultaneous presence of anti- PT antibodies is also associated with a higher risk of thrombotic events in paps patients. 18 Annexin V is a natural anticoagulant with a high calcium-dependent binding affinity for negatively charged phospholipids, one that acts competitively with coagulation factors (inhibition of the PT complex). High levels of anti-anx V antibodies have been found in 20% to 30% of SLE patients, 11,19 21 but it is not yet clear whether they are associated with a greater risk of thrombosis. However, these antibodies present an unusual characteristic that is of great interest in the obstetric field: of the various antiphospholipid and anti-cofactor antibodies, anti-anx V antibodies proved to be the greatest, if not the only, risk factor associated with recurrent pregnancy loss (RPL) in women with SLE 11 and in women without autoimmune disease or other risk factors. 22,23 A possible explanation is that, at least in murine models, the cofactor is needed to maintain the integrity of the placental structure and probably performs a thromboregulatory action at the maternalfetal interface. 24 Detection of anti-phospholipid cofactor antibodies in addition to the classic acl and LA antibodies therefore seems to be of considerable clinical importance. However, the number and heterogeneity of the target antigens of apl antibodies, and the fact that the apls can belong to various immunoglobulin classes, would require the performance of 10 to 12 different tests to provide a complete investigation and to obtain the maximum sensitivity, which would entail an unacceptable cost-benefit ratio. Hence, the practical need to choose the antibodies with the greatest diagnostic accuracy (ie, sensitivity and specificity) and to avoid the use of tests the results of which, however accurate, are very similar and have the same clinical significance. In a case-control study, we determined the prevalence and association with clinical manifestations of several apl and apl-cofactor antibodies in patients with paps, SLE, and in women with RPL, in order to establish whether their inclusion in the panel of apl tests would increase diagnostic sensitivity and specificity in cases in which acl antibodies were absent. MATERIALS AND METHODS The study population consisted of 311 patients: 25 were aclpositive with paps (3 men, 22 women; mean age 45 years, range years), all fulfilling the Sapporo criteria 1 ; 89 with SLE (16 men, 73 women; mean age 36 years, range years), classified according to the revised criteria of the American College of Rheumatology 2 ; and 77 with unexplained RPL (2 5 spontaneous miscarriages between the 8th and the 22nd week of gestation, without evidence of genetic, hormonal, or malformative causes; mean age 35 years, range years). Women with miscarriages only occurring before the 8th week were not included in the study because pregnancy loss before the 8th week is mostly caused by fetal chromosomal abnormality. 25,26 The presence of an acute or chronic infectious disease was ruled out in all patients. One hundred twenty healthy blood donors, matched for age and sex (94 women, 26 men; mean age 39 years, range years), were included as controls. Of the 89 patients with SLE, 23 had experienced a prior thrombotic episode (SLE/APS), whereas 66 had experienced no complications of this kind; of the 73 women with SLE, 4 had suffered a miscarriage before the diagnosis of SLE, and none had experienced one after the diagnosis. Blood donors who had suffered miscarriages were excluded from the control group. Informed consent was obtained from all patients and controls. Immunoglobulin (Ig) G and IgM acl, aapl (a mixture of anionic phospholipids: cardiolipin, phosphatidylserine, phosphatidyl inositol, and phosphatidic acid), anti- 2 GPI, anti-pt, and IgG anti-anx V antibodies were determined in all sera. The tests were performed using commercially available enzyme-linked immunosorbent assays (Orgentec Diagnostika GmbH, Mainz, Germany), according to the manufacturer s instructions. The assays were performed on microtiter plates coated with purified bovine cardiolipin for acl and purified bovine anionic phospholipids for aapl, both saturated with human 2 GPI. Anti- 2 GPI testing was performed using highly purified human antigen on -irradiated polystyrene microplates. Purified human antigens were employed for the anti-pt and anti-anx V assays. A polyclonal rabbit anti-human IgG (or IgM) conjugate, labeled with horseradish peroxidase, and a 3,3,5,5-tetramethylbenzidine enzymatic substrate were used in all assays. In every assay, antibody concentration was calculated using a 6-point calibration curve. Assays for acl were standardized using sera calibrated against the International Reference Material, 27 and the results were reported as anti-phospholipid IgG (GPL) or IgM (MPL) units. Anti- APL, anti- 2 GPI, anti-pt, and anti-anx V antibody levels were expressed in U/mL. All tests were performed in one laboratory by a single operator blinded to the patients clinical diagnosis, using the same batches for each antibody assay. All assays were validated by the inclusion of a positive and a negative control, as an internal quality control. Calibrators and controls were run in duplicate and patients samples in single determinations. Receiver operating characteristic curves were used to determine optimum cut-off levels for each antibody: they were set at 13.3 GPL and 10.6 MPL for acl; 10 U/mL for IgG and 12.5 U/mL for IgM aapl; 13.6 U/mL for IgG and 11.9 U/mL for IgM anti- 2 GPI; 13.7 U/mL for IgG and 10 U/mL for IgM anti-pt; and 5 U/mL for IgG anti-anx V. Statistical Analysis The diagnostic sensitivity and specificity of the acl, aapl, anti- 2 GPI, anti-pt, and anti-anx V antibodies were calculated in each disease group and in the healthy controls. The comparisons between patients and controls and between SLE patients with and without thrombosis were performed by means of the Mann-Whitney U test. Associations between antibody occurrence and a history of thrombosis were assessed with Fisher exact test. The Kruskall-Wallis analysis of variance test was used to compare antibody concentrations in the different groups, and a post hoc Tukey test was used to perform a posteriori multiple comparisons. Antibody concentrations are expressed as mean SD, with 95% confidence intervals (). Statistical analyses were performed using the SPSS 11.5 package for Windows (Chicago, Ill) and Prism 3.0 software for Windows (GraphPad Software, San 62 Arch Pathol Lab Med Vol 129, January 2005 Anti-Phospholipid Binding Protein Antibodies Bizzaro et al

3 Table 1. paps SLE RPL Healthy subjects Number and Percentage of Positivity for the Various Anti-Phospholipid and Anti-Cofactor Antibodies in the Disease Groups and Healthy Controls* acl Positive aapl Positive Anti- 2 GPI Positive Anti-PT Positive Anti-Anx V Positive IgG/IgM, IgG/IgM, IgG/IgM, IgG/IgM, No. No. (%) No. (%) No. (%) No. (%) No. (%) (100) 33 (37) 5 (6) 25 (100) 42 (47) 9 (12) 20 (80) 31 (35) 5 (6) 15 (60) 40 (45) 12 (16) 9 (8) 6 (24) 12 (13) 13 (17) * acl indicates anticardiolipin; Ig, immunoglobulin; aapl, anti anionic phospholipid antibodies; 2 GPI, 2 -glycoprotein I; PT, prothrombin; Anx V, annexin V; paps, primary anti-phospholipid syndrome; SLE, systemic lupus erythematosus; and RPL, recurrent pregnancy loss. Table 2. Percent Positivity of Anti-Cofactor Antibodies, Combined or as the Sole Antibody Present (in Parentheses), Broken Down by Disease Groups, in Anticardiolipin (acl)/anti Anionic Phospholipid Antibodies (aapl) Negative Patients* paps SLE RPL acl/aapl Negative, No Anti- 2 GPI Positive IgG/IgM, % 0 (0) 0 (0) Anti-PT Positive IgG/IgM, % 0 (0) 23 (14) 11 (8) Anti-Anx V Positive % 0 (0) 9 (5) 19 (16) * 2 GPI indicates 2 -glycoprotein I; PT, prothrombin; Anx V, annexin V; Ig, immunoglobulin; paps, primary anti-phospholipid syndrome; SLE, systemic lupus erythematosus; and RPL, recurrent pregnancy loss. Diego, Calif). Two-sided P values of less than.05 were considered to indicate statistical significance. RESULTS Prevalence of apl and apl-cofactor Antibodies in Patients With APS and SLE The prevalence rates of the various antibodies detected in the different groups are detailed in Table 1. Immunoglobulin G and/or IgM aapl, anti- 2 GPI, anti-pt, and IgG anti-anx V antibodies were detected in 25 (100%), 20 (80%), 15 (60%), and 6 (24%), respectively, of the 25 aclpositive paps patients; IgG and/or IgM acl, aapl, anti- 2 GPI, anti-pt, and IgG anti-anx V antibodies were detected in 33 (37%), 42 (47%), 31 (35%), 40 (45%), and 12 (13%), respectively, of the 89 SLE patients. The prevalence in the control group was 4% (96% specificity) for acl, aapl, anti- 2 GPI, and anti-anx V and 8% (92% specificity) for anti-pt. To evaluate whether the determination of anti-cofactor antibodies provides additional information to acl and aapl by increasing the diagnostic sensitivity of the antibody test, we analyzed the prevalence of anti- 2 GPI, anti- PT, and anti-anx V antibodies, both in association with one another and as the sole antibody present, in patients who were acl and aapl negative. In the 56 acl and aapl-negative SLE patients, the prevalence rates for anticofactor antibodies were 5% (4% alone) for anti- 2 GPI, 23% (14% alone) for anti-pt, and 9% (5% alone) for anti- Anx V (Table 2). Twenty-eight of 89 SLE patients tested negative for all the antibodies examined. In the remaining 61 patients who had at least one positive antibody, the percentage distribution demonstrated that acl antibodies were present in 54% of cases (5% alone), anti- 2 GPI in 50% of cases(3% alone), anti-pt in 65% of cases (26% alone), and anti-anx V in 20% of cases (5% alone) (Figure 1). Associations between the various apl and apl-cofactor antibodies and a history of thrombosis were evaluated in all patients with SLE. In the subset of 23 SLE/APS patients, IgG anti-pt prevalence was higher than that of the other autoantibodies (87% vs 70% acl, 66% aapl, 57% anti- 2 GPI, and 4% anti-anx V), and in 26% of cases, it was the only antibody present (Figure 2). Comparing the results of anti-pt and acl for sensitivity and specificity, anti-pt showed a higher sensitivity (87% vs 69.6%) and a slightly lower specificity (46% vs 49%) than acl. If the 2 antibodies were combined together, in patients with one or the other of the 2 antibodies (anti-pt or acl) and in patients with both antibodies (anti-pt and acl), the sensitivity would rise from 87% for anti-pt alone to 100% when either of the 2 antibodies is present, and the specificity would rise to 92.4% if both antibodies are detected (Table 3). Furthermore, in SLE patients, IgG anti-pt antibodies were associated with the highest risk of thrombosis (OR 15.3, P.001, vs 6.5 acl, 3.5 aapl, 3.4 anti- 2 GPI, 0.2 anti-anx V) (Table 4). Prevalence of apl and apl-cofactor Antibodies in Patients With RPL In women with RPL, the prevalence of IgG and/or IgM acl, aapl, anti- 2 GPI, anti-pt, and anti-anx V antibodies was 6%, 12%, 6%, 16%, and 17%, respectively (Table 1). In the 67 patients who tested negative for acl/aapl, anti- PT antibodies were present in 11% of cases (8% alone) and anti-anx V in 19% of cases (16% alone); no sample that was negative for acl or aapl proved to contain anti- 2 GPI antibodies (Table 2). Fifty-one of 77 patients tested negative for all the antibodies examined. In the remaining 26 patients who had at least one positive antibody, the percentage distribution demonstrated that acl antibodies were present in 19% of cases (4% alone), anti- 2 GPI in 19% of cases (4% alone), anti-pt in 46% of cases (27% alone), and anti-anx V in 50% of cases (42% alone) (Figure 3). Clinical Correlation of Antibody Concentrations The average antibody concentration, SD, and 95% for each of the antibodies considered, broken down by immunoglobulin class and disease group, are set out in Table Arch Pathol Lab Med Vol 129, January 2005 Anti-Phospholipid Binding Protein Antibodies Bizzaro et al 63

4 5. The mean levels of IgG and IgM acl, aapl, anti- 2 GPI, and IgG anti-pt were only significantly elevated (P.001) compared with similar values in the control group in the patients with thrombosis (paps and SLE/APS), whereas the anti-pt IgM values always gave results below the cut-off value and similar to those of the control group. The mean antibody concentration of IgG anti-anx V was only significantly increased (P.02) in patients with RPL. A statistically significant difference was observed between the mean levels of anti-anx V found in the 13 anti-anx V positive women in the RPL group (38.6 U/mL; SD, 47.5; range, ;, ) and in the anti-anx V positive patients, all of whom were women, in the other groups: 11.5 U/mL (SD, 8.2; range, ;, ; P.03) in the 6 women with APS; 18.0 U/mL (SD, 9.0; range, ;, ; P.04) in the 12 women with SLE; and 10.2 U/mL (SD, 9.1; range, ;, ; P.02) in the 5 women in the control group. It is noteworthy that of the 3 women with SLE who had anti- Anx V antibodies as the sole positive antibody, 2 had already had a miscarriage. Figure 1. Venn diagram showing percentage distribution of antibodies in the 61 patients with systemic lupus erythematosus (SLE) who had at least one positive antibody. The cumulative rates were 54% for anticardiolipin (acl), 51% for anti 2 -glycoprotein I (anti- 2 GPI), 65% for anti-prothrombin (anti-pt), and 20% for anti-annexin V (anti- Anx V). Figure 2. Venn diagram showing percentage distribution of antibody positivity in the 23 patients with systemic lupus erythematosus (SLE) and thrombosis. The overall positivity was 70% for anticardiolipin (acl), 57% for anti 2 -glycoprotein I (anti- 2 GPI), 87% for anti-prothrombin (anti-pt), and 4% for anti-annexin V (anti-anx V). COMMENT Determination of apl antibodies is commonly used to diagnose APS, to evaluate the risk of thrombosis in patients with SLE, and to test for the causes of thrombotic events or recurrent miscarriages in apparently healthy subjects with no risk factors. To increase the diagnostic sensitivity of immunoassays in the cases in which apl antibodies are absent, it can be useful to test for the presence of other autoantibodies directed against the plasma proteins that are involved in the coagulation processes and that act as phospholipid cofactors, such as 2 GPI, PT, and Anx V. Although none of these antibodies is included among the APS classification criteria, and none of them possesses absolute specificity, as they can be found in 15% to 30% of patients suffering from various infectious diseases without thrombosis, 28 there is recent evidence that they may play an important pathogenetic role, at least in some cases The prevalence data are highly variable, however, and those relating to their clinical usefulness are controversial. 33,34 For this study, we selected 3 groups of patients with a well-defined clinical diagnosis: one group with paps who were acl positive; one with SLE, stratified by the presence or absence of thrombotic events; and one with RPL. In all patients and the control group, anti-cofactor antibodies (anti- 2 GPI, anti-pt, and anti-anx V) were measured, in addition to acl and anionic apl, in order to evaluate their diagnostic sensitivity and specificity in the absence of positivity for acl and to establish the practical usefulness of their inclusion in the diagnostic profile of patients with SLE and women with RPL. The prevalence of anti- 2 GPI antibodies proved lower than that of acl antibodies in the group of patients with APS (80% vs 100%) and the group with SLE (35% vs 37%), Figure 3. Venn diagram showing percentage distribution of antibody positivity in the 26 patients with recurrent pregnancy loss who had at least one positive antibody. The highest frequency was found for antiannexin V (anti-anx V) (50%), then for anti-prothrombin (anti-pt) (46%), anticardiolipin (acl) (19%), and anti 2 -glycoprotein I (anti- 2 GPI) (19%). 64 Arch Pathol Lab Med Vol 129, January 2005 Anti-Phospholipid Binding Protein Antibodies Bizzaro et al

5 Table 3. acl anti-pt acl or anti-pt acl and anti-pt Sensitivity and Specificity of Anticardiolipin (acl) and Anti-prothrombin (PT) Antibodies Alone and Combined Together, in Patients With 1 of the 2 Antibodies Present, and in Patients With Both Antibodies* Sensitivity in 23 SLE Patients With Thrombosis, Specificity in 66 SLE Patients Without Thrombosis, No. Positive (%) No. Positive (%) 16 (69.6) 20 (87.0) 23 (100) 13 (56.5) * The presence of acl or anti-pt provides a sensitivity of 100% for thrombosis. 17 (74.2) 20 (69.7) 32 (51.5) 5 (92.4) acl Table 4. Prevalence, Statistical Significance (P), and Odds Ratio (OR) of the Various Anti-Phospholipid and Anti- Phospholipid Cofactor Antibodies in 89 Patients With Systemic Lupus Erythematosus, in Relation to the Presence or Absence of Thrombotic Events aapl Test* Anti- 2 GPI Anti-PT Anti-Anx V Thrombosis Yes No P OR 95% * acl indicates anticardiolipin; aapl, anti anionic phospholipid antibodies; 2 GPI, 2 -glycoprotein I; PT, prothrombin; and Anx V, annexin V. Table 5. Mean Value of Antibody Concentration, SD, and 95% Confidence Interval () for Each of the Antibodies Considered, Broken Down by Immunoglobulin Class, in the Disease Groups and Healthy Controls* Cut-off paps Mean SD SLE/APS Mean SD 13.3 GPL acl 85.8* * SLE without thrombosis Mean 9.1 SD RPL Mean SD Healthy subjects Mean 6.4 SD IgM, 10.6 MPL 23.7* ** U/mL 73.9* * aapl IgM, 12.5 U/mL 33.5* *** U/mL 66.5* * Anti- 2 GPI IgM, 11.9 U/mL 25.4* **** U/mL 53.0* * Anti-PT IgM, 10 U/mL Anti-Anx V 5 U/mL ***** * P.001; ** P.005; *** P.002; **** P.003; ***** P.02. acl indicates anticardiolipin; aapl, anti anionic phospholipid antibodies; 2 GPI, 2 -glycoprotein I; PT, prothrombin; Anx V, annexin V; Ig, immunoglobulin; paps, primary anti-phospholipid antibody syndrome; SLE, systemic lupus erythematosus; and RPL, recurrent pregnancy loss. although anti- 2 GPI antibodies were present in 1 of the acl-negative patients with SLE and thrombosis. Similar findings to ours were obtained very recently by Theodoridou et al, 35 who found no positivity for anti- 2 GPI antibodies in 26 acl-negative patients with SLE and thrombosis, and by Hsieh et al, 36 who, in a study of 418 acl/ LA-negative patients with venous thromboembolism who did not suffer from autoimmune disease, only found anti- 2 GPI antibodies in 1.7% of cases and in only 66.7% of another 27 LA-positive patients. Both groups of authors concluded that determination of anti- 2 GPI antibodies does not improve the clinical management of patients with Arch Pathol Lab Med Vol 129, January 2005 Anti-Phospholipid Binding Protein Antibodies Bizzaro et al 65

6 a history of thrombosis without LA or acl. Consequently, at least on the basis of our results and those of these 2 studies, determination of anti- 2 GPI antibodies does not increase the diagnostic sensitivity of apl tests, and their recently proposed introduction 37,38 as an alternative screening test in substitution to the well-established acl tests remains questionable. However, since anti- 2 GPI antibodies have a higher specificity for the diagnosis of APS than the conventional acl test, 37,38 their use to confirm acl-positive results can be recommended. In SLE patients we found that anti-pt antibodies of the IgG class were more prevalent (45%) than acl (37%) or other anti-cofactor antibodies. IgG anti-pt were also positive in 87% of the SLE patients who had suffered a thrombotic event, whereas acl were positive in 69.6%. This means that in patients with SLE and SLE/APS, IgG anti- PT antibodies are those with the greatest sensitivity of all the various autoantibodies directed against the phospholipids or their cofactors, confirming the results of a recent study by Nojima et al, 15 which demonstrated the presence of anti-pt antibodies in 46% of SLE patients for whom, together with LA, they constituted the only risk factor for venous thromboembolism. Numerous other researchers have found an association between anti-pt and thrombosis. Puurunen et al 39 reported the presence of anti-pt in 34% of patients with SLE and found a positive correlation with deep vein thrombosis in this population. Bertolaccini et al 40 reported positivity for anti-pt in 58 of 207 patients with SLE and observed that 53% had a history of thrombotic events. Furthermore, in a series of 177 patients with SLE or paps, Muñoz-Rodriguez et al 14 found that thrombotic events were more prevalent in patients with anti-pt and that these antibodies represented an independent risk factor for thrombosis. In a study of patients without autoimmune disease, Vaarala et al 41 showed that high levels of anti-pt conferred a 2.5-fold increase in the risk of myocardial infarction in middle-aged men, and Palosuo et al, 17 who also studied 265 middle-aged men, found a close relationship between high anti-pt levels and deep vein thrombosis and pulmonary embolia. On the other hand, Horbach et al, 42 who studied 175 patients with SLE, found that both IgG and IgM anti-pt were more frequent in patients with a history of thrombosis, but the correlation was not significant when examined by a multivariate analysis. Pengo et al 43 and Swadzba et al 44 also found no correlation between the presence of anti-pt and thrombosis in patients with APS or with SLE. In this study, we found that although acl, anti- 2 GPI, and aapl were significant risk factors for thrombosis in SLE, the highest risk was associated with anti-pt antibodies. The best results in terms of diagnostic accuracy were obtained by combining together acl and anti-pt; actually, the presence of one or the other antibodies brings the sensitivity for thrombosis to 100%, and their simultaneous positivity increases the specificity to 92.4%. It is interesting to note that in SLE patients, the sensitivity of the aapl test that contained a mixture of anionic phospholipids was greater than that of acl tests (47% vs 37%). As the antigens used in the aapl test included cardiolipin, it can be deduced that 10% of SLE sera contained antibodies directed against one of the other anionic phospholipids: phosphatidylserine, phosphatidyl inositol, or phosphatidic acid. However, none of the aapl-positive, acl-negative patients with SLE belonged to the subgroup with thrombosis, so these antibodies do not seem to have any greater clinical significance than acl. With regard to the group with RPL, we found anti-pt antibodies in 16% of the women with RPL, but we could not demonstrate a significant association with miscarriage; this conflicts with the findings of some researchers 12 but agrees with those of others. 45 Differences in the reported prevalence and association with clinical manifestations of anti-pt antibodies may reflect patient selection, antibody heterogeneity, and their different affinity for human PT, 46 as well as different antigenic substrate composition (phosphatidylserine-bound PT or PT alone 47,48 ), or methodological variation (-irradiation of microplates, coating procedure, calibration, and choice of cut-off levels 11,43,47 ). Until an international reference preparation and standard analysis methods are available, it is particularly difficult to perform a correct comparative evaluation of the results obtained in the various studies and to define the clinical significance of anti-pt antibodies in women with RPL. Although Donohoe and colleagues 49 reported high levels of anti-pt antibodies of the IgM class in 61% of paps and in 59% of SLE patients, and although they found that only IgM and not IgG anti-pt were associated with thrombosis, in our study none of the patients with paps or SLE and thrombosis tested positive for IgM anti-pt. Similar results to ours were obtained by Nojima et al. 15 However, since very few studies have been performed to date on IgM anti-pt, it is still uncertain whether testing IgM anti-pt in association with IgG anti-pt would improve the sensitivity for thrombosis compared to testing for IgG alone. In the group of patients with RPL, anti-anx V antibodies were present in 17% of cases; this was the only serologic parameter significantly associated with miscarriage, which confirmed the results of other studies. 11,22,23 In particular, Satoh et al 50 and Kaburaki et al 51 found a significantly higher frequency of thrombosis or fetal loss in patients with SLE/APS and anti-anx V IgG positivity, and Gris et al 52 found that anti-anx V antibodies were an independent retrospective risk factor for unexplained early fetal loss. Conversely, although Arnold et al 53 found anti- Anx V antibodies in 35% of the acl-positive and 19% of the acl-negative women who had experienced miscarriages, he did not find that anti-anx V constituted a risk factor for miscarriage; equally, Ogawa et al 21 found no association between Anx V and miscarriage in patients with APS. In our patients, neither acl, anti- 2 GPI, nor aapl antibodies proved to be associated with miscarriage. Other researchers have found no link between acl 22,54 or anti- 2 GPI 52,54,55 and miscarriages, whereas in a prospective study of 60 patients with SLE, Cortés-Hernández et al 56 found that both acl and anti- 2 GPI antibodies were associated with miscarriage. Taken as a whole, these findings seem highly contradictory, and in our opinion, it is not yet possible to evaluate whether anti-anx V antibodies, or other antibodies among the various apls, are a useful parameter to define the risk of miscarriage. In conclusion, the results of this study indicate that anti- PT antibodies are a very sensitive marker associated with thrombosis in SLE patients. Specifically, anti-pt could detect 17% of patients with SLE and thrombosis who were negative for acl, and the combined measurement of acl and anti-pt could provide a 100% sensitivity for thrombosis. Therefore, their measurement can be usefully as- 66 Arch Pathol Lab Med Vol 129, January 2005 Anti-Phospholipid Binding Protein Antibodies Bizzaro et al

7 sociated with the more widely used acl antibodies in the prognostic evaluation of SLE patients. In this series, anti-anx V was the only antibody associated with RPL, and although conflicting results are present in the literature, the majority of studies seem to indicate a possible association with recurrent miscarriage. However, prospective studies are needed to clarify whether each antibody is not only associated with, but also predictive of, the development of thrombotic complications. We thank Orgentec Diagnostika (Mainz, Germany) and Bouty Laboratories (Milan, Italy) for providing reagents free of charge. References 1. Wilson WA, Gharavi AE, Koike T, et al. International consensus statement on preliminary classification criteria for definite antiphospholipid syndrome. Arthritis Rheum. 1999;42: Hochberg MC. The American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1997;40: Galli M, Comfurius P, Maassen C, et al. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet. 1990;335: 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: beta 2 -glycoproyein I (apolipoprotein H). Proc Natl Acad Sci U S A. 1990;87: Simmelink MJA, Horbach DA, Derksen RHWM, et al. Complexes of antiprothrombin antibodies and prothrombin cause lupus anticoagulant activity by competing with the binding of clotting factors for catalytic phospholipid surfaces. Br J Haematol. 2001;113: Rao LVM, Hoang AD, Rapaport SI. Mechanism and effects of the binding of lupus anticoagulant IgG and prothrombin to surface phospholipid. Blood. 1996;88: Zhao Y, Rumold R, Zhu M, et al. An IgG antiprothrombin antibody enhances prothrombin binding to damaged endothelial cells and shortens plasma coagulation times. Arthritis Rheum. 1999;42: Rand JH, Wu X, Guller S, Scher J, Andree HAM, Lockwood CJ. Antiphospholipid immunoglobulin G antibodies reduce annexin V levels on syncitiotrophoblast atypical membranes and in culture media of placental villi. Am J Obstet Gynecol. 1997;177: Koike T, Matsuura E. Anti-beta 2 glycoprotein I antibody: specificity and clinical significance. Lupus. 1996;5: Cucurull E, Espinoza LR, Mendez E, et al. Anti-cardiolipin and anti- 2 glycoprotein-1 antibodies in patients with systemic lupus erythematosus: comparison between Colombians and Spaniards. Lupus. 1999;8: Nojima J, Kuratsune H, Suehisa E, et al. Association between the prevalence of antibodies to 2 -glycoprotein I, prothrombin, protein C, protein S, and annexin V in patients with systemic lupus erythematosus and thrombotic and thrombocytopenic complications. Clin Chem. 2001;47: von Landenberg P, Matthias T, Zaech J, et al. Antiprothrombin antibodies are associated with pregnancy loss in patients with the antiphospholipid syndrome. Am J Reprod Immunol. 2003;49: Forastiero RR, Martinuzzo ME, Cerrato GS, Kordich LC, Carreras LO. Relationship of antibeta 2 -glycoprotein I and antiprothrombin antibodies to thrombosis and pregnancy loss in patients with antiphospholipid antibodies. Thromb Haemost. 1997;78: Muñoz-Rodriguez FJ, Reverter JC, Font J, et al. Prevalence and clinical significance of antiprothrombin antibodies in patients with systemic lupus erythematosus or with primary antiphospholipid syndrome. Haematologica. 2000; 85: Nojima J, Kuratsune H, Suehisa E, et al. Anti-prothrombin antibodies combined with lupus anticoagulant activity is an essential risk factor for venous thromboembolism in patients with systemic lupus erythematosus. Br J Haematol. 2001;114: Pasquier E, Amiral J, de Saint Martin L, Mottier D. A cross sectional study of antiphospholipid-protein antibodies in patients with venous thromboembolism. Thromb Haemost. 2001;86: Palosuo T, Virtamo J, Haukka J, et al. High antibody levels to prothrombin imply a risk of deep venous thrombosis and pulmonary embolism in middle-aged men. Thromb Haemost. 1997;78: Lakos G, Kiss E, Regeczy N, et al. Anti-prothrombin and antiannexin V antibodies imply risk of thrombosis in patients with systemic autoimmune diseases. J Rheumatol. 2000;27: Nakamura N, Kuragaki C, Shidara Y, Yamaji K, Wada Y. Antibody to annexin V has antiphospholipid and lupus anticoagulant activity. Am J Hematol. 1995;49: Matsuda J, Saitoh N, Gohchi K, Gotoh M, Tsukamoto M. Anti-annexin V antibody in systemic lupus erythematosus patients with lupus anticoagulant and/ or anticardiolipin antibody. Am J Hematol. 1994;47: Ogawa H, Zhao D, Dlott JS, et al. Elevated antiannexin V antibody levels in antiphospholipid syndrome and their involvement in antiphospholipid antibody specificities. Am J Clin Pathol. 2000;114: Matsuda J, Gotoh M, Saitoh N, Gohchi K, Tsukamoto M, Yamamoto T. Antiannexin antibody in the sera of patients with habitual fetal loss or preeclampsia. Thrombosis Res. 1994;75: Matsubayashi H, Arai T, Izumi S, Sugi T, McIntyre JA, Makino T. Antiannexin V antibodies in patients with early pregnancy loss or implantation failure. Fertil Steril. 2001;76: Wang X, Campos B, Kaetzel MA, Dedman JR. Annexin V is critical in the maintenance of murine placental integrity. Am J Obstet Gynecol. 1999;180: Molo MW, Kelly M, Balos R, Mullaney K, Radwanska E. Incidence of fetal loss in patients after detection of fetal heart activity with early transvaginal ultrasound. J Reprod Med. 1993;38: Regan L, Rai R. Epidemiology and the medical causes of miscarriage. Bailliere s Clin Obstet Gynaecol. 2000;14: Harris EN, Hughes GR. Standardising the anticardiolipin antibody test. Lancet. 1987;1: Loizou S, Singh S, Wypkema E, Asherson RA. Anticardiolipin, anti- 2 -glycoprotein I and antiprothrombin antibodies in black South African patients with infectious disease. Ann Rheum Dis. 2003;62: Haj-Ichya S, Sherer Y, Blank M, Shoenfeld Y. apts induce thrombosis in an animal model of APS. Lupus. 2002;11: Arnout J, Boon D, Vandervoort P, Vanrusselt M, Vermylen J. A monoclonal antibody against prothrombin fragment 2 behaves like a lupus anticoagulant. Lupus. 2002;11: Rote NS, Kumar N, Chang SH, Liln L, Xu B, Kent MN. Monoclonal antibody against annexin V prevents intertrophoblast fusion. Lupus. 2002;11: Ornoy A, Tartakover-Matalon S, Yacobi S, Blank M, Blumenfeld Z, Shoenfeld Y. The effects of IgG purified from women with SLE/APS and associated pregnancy loss on rat embryos in-vitro. Lupus. 2002;11: Galli M, Barbui T. Antiprothrombin antibodies: detection and clinical significance in the antiphospholipid syndrome. Blood. 1999;93: Galli M. Which antiphospholipid antibodies should be measured in the antiphospholipid syndrome? Haemostasis. 2000;30: Theodoridou A, Bertolaccini M, Hamid C, Khamashta MA, Hughes GRV. The value of testing for antiphospholipid antibodies, other than acl and LA, in systemic lupus erythematosus patients with thrombosis. Arthritis Rheum. 2002; 46(suppl):S Hsieh K, Knöbl P, Rintelen C, et al. Is the determination of antibeta 2 glycoprotein I antibodies useful in patients with venous thromboembolism without the antiphospholipid syndrome? Br J Haematol. 2003;123: Roubey RAS, Maldonado MA, Byrd SN. Comparison of an enzyme-linked immunosorbent assay for antibodies to 2 -glycoprotein I and a conventional anticardiolipin immunoassay. Arthritis Rheum. 1996;39: Amengual O, Atsumi T, Khamashta MA, Koike T, Hughes GRV. Specificity of ELISA for antibody to 2 -glycoprotein I in patients with antiphospholipid syndrome. Br J Rheumatol. 1996;35: Puurunen M, Vaarala O, Julkunen H, Aho K, Palosuo T. Antibodies to phospholipid-binding plasma proteins and occurrence of thrombosis in patients with systemic lupus erythematosus. Clin Immunol Immunopathol. 1996;80: Bertolaccini ML, Atsumi T, Khamashta MA, Amengual O, Hughes GR. Autoantibodies to human prothrombin and clinical manifestations in 207 patients with systemic lupus erythematosus. J Rheumatol. 1998;25: Vaarala O, Puurunen M, Manttari M, Manninen V, Aho K, Palosuo T. Antibodies to prothrombin imply a risk of myocardial infarction in middle-aged men. Thromb Haemost. 1996;75: Horbach DA, van Oort E, Donders RC, Derksen RH, de Groot PG. Lupus anticoagulant is the strongest risk factor for both venous and arterial thrombosis in patients with systemic lupus erythematosus. Comparison between different assays for the detection of antiphospholipid antibodies. Thromb Haemost. 1996;76: Pengo V, Biasiolo A, Brocco T, Tonetto S, Ruffatti A. Autoantibodies to phospholipid-binding plasma proteins in patients with thrombosis and phospholipidreactive antibodies. Thromb Haemost. 1996;75: Swadzba J, De Clerck LS, Stevens WJ, et al. Anti-cardiolipin, antibeta(2)- glycoprotein I, antiprothrombin antibodies, and lupus anticoagulant in patients with systemic lupus erythematosus with a history of thrombosis. J Rheumatol. 1997;24: Forastiero R, Martinuzzo ME, Adamczuk Y, Carreras LO. Occurrence of antiprothrombin and antibeta 2 -glycoprotein I antibodies in patients with a history of thrombosis. J Lab Clin Med. 1999;134: Amengual O, Atsumi T, Koike T. Specificities, properties, and clinical significance of antiprothrombin antibodies. Arthritis Rheum. 2003;48: Galli M, Beretta G, Daldossi M, Bevers EM, Barbui T. Different anticoagulant and immunological properties of antiprothrombin antibodies in patients with antiphospholipid antibodies. Thromb Haemost. 1997;77: Matsuda J, Saitoh N, Gotoh M, Kawasugi K, Gohchi K, Tsukamoto M. Phosphatidyl serine-dependent antiprothrombin antibody is exclusive to patients with lupus anticoagulant. Br J Rheumatol. 1996;35: Donohoe S, Mackie IJ, Isenberg D, Machin SJ. Anti-prothrombin antibodies: assay conditions and clinical associations in the antiphospholipid syndrome. Br J Haematol. 2001;113: Satoh A, Suzuki K, Takayama E, et al. Detection of antiannexinv antibodies Arch Pathol Lab Med Vol 129, January 2005 Anti-Phospholipid Binding Protein Antibodies Bizzaro et al 67

8 in patients with antiphospholipid syndrome and systemic lupus erythematosus. J Rheumatol. 1999;26: Kaburaki J, Kuwana M, Yamamoto M, Kawai S, Ikeda Y. Clinical significance of antiannexin V antibodies in patients with systemic lupus erythematosus. Am J Hematol. 1997;54: Gris JC, Quéré I, Sanmarco M, et al. Antiphospholipid and antiprotein syndromes in nonthrombotic, nonautoimmune women with unexplained recurrent primary early foetal loss. Thromb Haemost. 2000;84: Arnold J, Holems Z, Pickering W, Farmer C, Regan L, Cohen H. Antibeta 2 glycoprotein 1 and antiannexin V antibodies in women with recurrent miscarriage. Br J Haematol. 2001;113: Gregg L, Sibbitt WL, Patel S, Maldonado ME, Bankhurst AD. Predictors of fetal loss in patients with systemic lupus erythematosus. Arthritis Rheum. 2001; 44(suppl):S Ailus K, Tulppala M, Palosuo T, Ylikorkala O, Vaarala O. Antibodies to beta 2 glycoprotein I and prothrombin in habitual abortion. Fertil Steril. 1996;66: Cortés-Hernández J, Ordi-Ros J, Paredes F, Casellas M, Castillo F, Vilardell- Tarres M. Clinical predictors of fetal and maternal outcome in systemic lupus erythematosus: a prospective study of 103 pregnancies. Rheumatology. 2002;41: Arch Pathol Lab Med Vol 129, January 2005 Anti-Phospholipid Binding Protein Antibodies Bizzaro et al