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The clinical value of testing for antibodies to phosphatidylethanolamine (aPE) in patients with systemic lupus erythematosus (SLE)
Thrombosis Research 130 (2012) 914–918
Contents lists available at SciVerse ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Regular Article
The clinical value of testing for antibodies to phosphatidylethanolamine (aPE) in patients with systemic lupus erythematosus (SLE) M.L. Bertolaccini a,⁎, V. Murru a, S. Sciascia a, G. Sanna b, M.A. Khamashta a, b a b
Lupus Research Unit, The Rayne Institute, Division of Women's Health, King's College London, UK Louise Coote Lupus Unit, Guy's and St. Thomas’ NHS Foundation Trust, St. Thomas’ Hospital, London, UK
a r t i c l e
i n f o
Article history: Received 3 May 2012 Received in revised form 30 May 2012 Accepted 6 June 2012 Available online 15 October 2012 Keywords: Antibodies to phosphatidylethanolamine Antiphospholipid syndrome Thrombosis Pregnancy morbidity Systemic lupus erythematosus
a b s t r a c t The value of testing for aPE in venous thrombosis and fetal death is in constant debate. We evaluated if testing for aPE has a diagnostic value in patients with SLE. Patients and Methods: We included 224 patients. aPE were tested by an in-house ELISA using FCS. Results: aPE were found in 41% of the patients. IgG and IgM aPE were more frequently found along with other aPL than in those negative for aPL (p = 0.003 and p = 0.01). IgG aPE were more frequently found in patients with definite APS than in those without (p = 0.003). aPE were more frequent in patients with thrombosis than in those without, particularly the IgG isotype (p = 0.03). When subdividing between venous and arterial thrombosis, only an association between IgG aPE with venous thrombosis was retained (p = 0.01). Titres of IgG aPE were significantly higher in patients with arterial or those with venous thrombosis, when compared to the patients without thrombosis (p = 0.004 and p = 0.001). Titres of IgM aPE were higher in patients with arterial thrombosis when compared to those without (p = 0.014). No associations were found between the presence of aPE and/or pregnancy morbidity. The presence of aPE did not correlate with that of any other aPL. After multivariate analysis all clinical associations failed to retain significance. Conclusions: aPE are frequently seen in SLE and do not correlate with other routinely tested aPL. Although more prevalent, aPE is not an independent risk factor for thrombosis or pregnancy morbidity in patients with SLE. © 2012 Elsevier Ltd. All rights reserved.
Introduction The antiphospholipid syndrome (APS) is a common acquired thrombophilia characterised by vascular thrombosis and/or pregnancy morbidity in the presence of antiphospholipid antibodies (aPL). aPL are a heterogeneous group of autoantibodies whose specificity is directed to phospholipid-binding proteins or their complex with phospholipids. In clinical practice, anticardiolipin antibodies (aCL), antibodies to β2 glycoprotein I (anti-β2GPI) detected by ELISA and the lupus anticoagulant (LA) detected by clotting assays are the most widely used and standardised tests for the detection of aPL. However, a variety of plasma proteins have been implicated as targets for these antibodies. Phosphatidylethanolamine (PE) is a ubiquitous phospholipid present in both the internal and external sides of normal cell membranes [1]. Antibodies to PE (aPE) have been reported to be present on a ⁎ Corresponding author at: Lupus Research Unit, The Rayne Institute, Division of Women's Health, King's College London, 4th Floor Lambeth Wing, St. Thomas’ Hospital, London SE1 7EH, UK. Tel.: + 44 2071883569; fax: + 44 2076202658. E-mail address: [email protected] (M.L. Bertolaccini). 0049-3848/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2012.06.008
range from 17% [2] to 80% [3]. In most of the cases, aPE have been reported in association with other aPL [2,4,5]. However, few reports showed aPE in the absence of aCL and/or LA [6,7]. The presence of aPE have been associated with thrombosis [5–9] and recurrent pregnancy loss [10–12] but not all studies arrived to the same conclusions [13] and, therefore, screening for aPE is still not recommended. The purpose of the present study was to evaluate if testing for aPE has a diagnostic value in patients with systemic lupus erythematosus (SLE). Patients and methods Patients This study included 224 patients, all fulfilling the 1982 criteria for SLE [14]. Of these, 59 patients fulfil criteria for definite APS [15,16] and 55 were positive for aPL without fulfilling criteria. Patients group comprised 212 women with a mean age of 42.7 ± 11.9 years and mean disease duration of 12.2 ± 8.8 years. Overall, 81 patients had a history of thrombosis (34 arterial, 27 venous and 20 both arterial and venous thrombosis). Out of 143 women who had ever been pregnant, 39 had a history of miscarriages
M.L. Bertolaccini et al. / Thrombosis Research 130 (2012) 914–918
and 36 a history of fetal death. Demographics are summarised in Table 1. Ethical approval was obtained from the Guy's and St Thomas’ Ethics committee and all patients involved in this study gave their written consent. Methods Blood collection Blood was collected by venopuncture from the antecubital vein into pre-cooled tubes containing 0.105 M sodium citrate and in non-anticoagulated tubes (Hemogard® 9NC and Hemogard® Z, respectively, Becton Dickinson, Rutherford, USA). Platelet free plasma was obtained by centrifugation at 2500 g for 20 minutes and filtration using a 0.2 μm surfactant free cellulose acetate membrane (Nalgene, Rochester, NY, USA). Plasma was stored at −80 °C until used. For sera preparation, blood was allowed to clot at room temperature and then centrifuged at 2000 g for 20 minutes. Sera were subsequently aliquoted and stored frozen at − 80 °C until use. Antibodies to phosphatidylethanolamine (aPE) testing aPE were tested as described by SanMarco et al. [17] with some modifications. Briefly, microtiter plates were coated with 50 μg/ml of phosphatidylethanolamine in methanol/chloroform (4:1) overnight at 4 °C. After washing and blocking for 1 hour with 10% fetal calf serumPBS, 50 μl of samples diluted 1:100 were added and incubated for 2 hours. After washing with PBS, alkaline phosphatase-conjugated anti-human IgG and/or IgM were added in the appropriate dilution and incubated for a further hour. All incubations were done at room temperature. After 1 hour and 3 washes with PBS, 100 μl of 1 mg/ml of p-nitrophenylphosphate (Sigma) in 1 M diethanolamine buffer (pH 9.8) were added. Optical density at 405/620 nm was measured by a Titertek Multiskan MC apparatus (Flow Laboratories, Herts, UK) and converted to units (U) with a sample showing a high binding used as a standard. The cut-off value was established on 8 AU for IgG aPE and 9 AU for IgM aPE, determined by the 99th percentile of 140 apparently healthy donors.
915
Anticoagulant-Phospholipid-dependent antibodies [20], using the Automated Coagulation Laboratory (ACL) 300R (Instrumentation Laboratory, Milan, Italy). All samples were screened using the activated partial thromboplastin time (aPTT–IL test™ APTT-SP, Instrumentation Laboratory, Italy). The dilute Russell viper venom time (dRVVT) coagulation test was performed as described by Thiagarajan et al. [21]. Antibodies to prothrombin were tested by the aPT (solid phase) and aPS/PT (phosphatidylserine-prothrombin complex) as previously reported [22,23]. Statistical analysis All statistical analysis was performed using SPSS 16.0 program (Microsoft software). Categorical analysis was expressed as odds ratio with its 95% confidence interval (OR [95%CI]), where a lower limit >1.0 was considered significant. The degree of linear association between aPE and other aPL was quantified by the Spearman's correlation method. Quantitative data were analysed by the Mann–Whitney test. All p values were determined by Fisher's exact test. A stepwise forward conditional procedure including all the variables used for the univariate analysis was used for the logistic regression analysis to explore the value of aPE. A p value of b0.05 was considered statistically significant. Results aPE were present in 92 (41%) patients. Of these, 68 had IgG aPE and 13 IgM aPE and 11 had both, IgG and IgM aPE. Prevalence of all aPL tested is shown in Table 2. IgG and IgM aPE were more frequently found in patients with aPL (n = 114) than in those without aPL (46% vs. 23%, OR 2.8 [95%CI 1.5–4.9], p = 0.003 and 15% vs. 5%, OR 3.2 [95%CI 1.2–8.5], p = 0.01, respectively). However, only IgG aPE were more frequently found in patients with definite APS than in those without (50% vs. 29%, OR 2.4 [95%CI 1.3–4.5], p = 0.003). Relationship of aPE to thrombosis and pregnancy morbidity
Other aPL The aCL ELISA was performed according to the standardised technique [18]. Anti-β2GPI were detected by ELISA as described previously [19], using irradiated microtitre plates (Nunc Maxisorp, Denmark). Plasma samples were tested for the presence of LA according to the recommended criteria from the ISTH Subcommittee on Lupus Table 1 Demographic characteristics of SLE. SLE n = 224 (%) Female (%) Mean age ± SD Mean disease duration ± SD APS* aPL (no APS) † Thrombosis‡ –Arterial thrombosis –Venous thrombosis Pregnancy morbidity§ –Miscarriages (≥1) –Miscarriages (≥3) –Fetal death
212 (95) 42.7 ± 11.9 12.2 ± 8.8 59 (26) 55 (24) 81 (36) 54 (24) 47 (20) 40 (27) 39 (27) 9 (6) 36 (25)
SLE: systemic lupus erythematosus, APS: antiphospholipid syndrome, aPL: antiphospholipid antibodies, including aCL, LA and/or anti-β2GPI. * All patients fulfilled criteria for APS [15,16]. †aPL included patients who were positive for aPL but did not fulfilled criteria (i.e. low titres and/or no clinical events attributable to APS). ‡20 patients from each group have both arterial and venous thrombosis. §Pregnancy morbidity was defined by APS criteria (ref). All pregnancy data percentages calculated over the total number of female who had ever been pregnant (n = 143).
Only IgG aPE were more frequently found in patients with thrombosis than in those without (44% vs. 30%, OR1.8 [95%CI 1.0–3.2], p = 0.03). When subdividing the thrombosis group between those with venous and those with arterial thrombosis, only the association between IgG aPE with venous thrombosis was retained (51% vs. 31%, OR2.3 [95%CI 1.2–4.4], p = 0.01). Median titres [range] of IgG aPE were significantly higher in patients with thrombosis as a whole and in those with arterial or those with venous, when compared to the patients without thrombosis (7.35 AU [1.5–81.5] and 7.23 AU [2.4–42.1] or 8.16 [1.5–81.5] vs. 5.85 AU [0.7–50.2], p = 0.004, p = 0.004 and p = 0.001 respectively). Titres of IgM aPE were higher in patients with thrombosis, particularly arterial thrombosis when compared to those without (2.0 AU [0–359.7] and 2.35 AU [0.1–359.7] vs. 1.5 AU [0–129.5], p = 0.022 and p = 0.014 respectively). All data on aPE titres is summarised in Fig. 1. No associations were found between the presence of aPE and/or pregnancy morbidity, miscarriages or fetal death in this cohort. Prevalence of aPE in women with available obstetric history is depicted in Table 3. aPE did not correlate with any other aPL including aCL and antiβ2GPI (Fig. 2) or aPT, aPS/PT and LA (data not shown). Multivariate analysis Multivariate analysis was used to examine the association of aPE to thrombosis and or pregnancy morbidity in patients with SLE after accounting for potential confounders, including other aPL, age and gender.
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Table 2 Prevalence of aPE and other aPL in SLE. aPL ⁎
SLE n = 224 (%)
aPE IgG/IgM –aPE IgG –aPE IgM aCL IgG/IgM –aCL IgG –aCL IgM Anti–β2GPI IgG/IgM –Anti-β2GPI IgG –Anti-β2GPI IgM aPT IgG/IgM –aPT IgG –aPT IgM aPS/PT IgG/IgM –aPS/PT IgG –aPS/PT IgM LA
92 79 24 126 111 46 48 38 14 68 57 15 68 55 33 56
(41) (35) (10) (56) (49) (20) (21) (16) (6) (30) (25) (6) (30) (24) (14) (25)
⁎ some patients were positive for more than 1 antibody and/or isotype. IgG/M: IgG and/or IgM. aPE: anti-phosphatidylethanolamine; aCL: anticardiolipin antibodies; anti-β2GPI: antibodies to β2 glycoprotein I, aPT: antibodies to prothrombin in solid phase; aPS/PT: antibodies to phosphatidylserine-prothrombin complex; LA: lupus anticoagulant.
After multivariate analysis, all clinical associations failed to retain significance (Table 4). Discussion In this study we attempted to analyse the value of testing for aPE as a marker of risk for thrombosis in a large cohort of patients with SLE. As aPE appear to be markers of risk for thrombosis, we evaluated the prevalence and clinical significance of aPE in a large number of patients with SLE. In this study we found an overall prevalence of aPE of 41%, with IgG and IgM isotypes present in 35% and 10% of the patients, respectively. There was a correlation between the presence of aPE and the clinical manifestations of APS and the relevance of these antibodies as markers for APS is discussed. We found that aPE are frequently found in patients with SLE, particularly in those with aPL. Although frequently seen in association with other aPL, aPE did not correlate with aCL, anti-β2GPI, aPT, aPS/ PT and/or the LA, suggesting that they are a different subpopulation of aPL. Few studies available in the literature investigated the clinical significance of aPE. The presence of aPE has been associated with thrombosis since the early 90's [5–7]. In 1998, we studied aPE in a large cohort of SLE [24]. At the time, we reported a low prevalence of aPE, now attributed to differences in the protocol used and no associations between aPE and clinical features of APS. It has recently being reported that aPE reactivity is dependent on the lipid concentration of the buffer component [17]. Whilst in our 1998 study, we used adult bovine serum (rich in lipid content), in the present study we used fetal calf serum with the consequent improvement in aPE detection. In 2007, a multicenter study followed [8] reporting IgG aPE to be an independent risk factor for venous thrombosis with an odds ratio of 6. In our present study, we found an association between the presence of aPE and the occurrence of vascular events. When patients with thrombosis were subdivided into arterial events and venous events, only IgG aPE remained associated with venous thrombosis. However, after multivariate analysis, the association between aPE and venous thrombosis failed to retain significance. In contrast, the prevalence of IgM aPE was not different between patients with arterial thrombosis and venous thrombosis.
Fig. 1. Distribution of IgG (a) and IgM (b) aPE in SLE with and without thrombosis.
aPE have been reported to be associated with pregnancy morbidity and 2 different studies have shown aPE to be a stronger independent risk factor for early miscarriages when compared to aCL and anti-β2GPI [11,12]. Interestingly, aPE have also been described as the only aPL found in most of the cases (73%). In our study, aPE were not associated to pregnancy morbidity, miscarriages or fetal death. These contradictory results can be due to the lack of consensual standardized method for the measurement of aPE and the Table 3 Prevalence of aPE in 143 women with available obstetric history. aPE
IgG/M n = 63 IgG n = 56 IgM n = 40
Pregnancy morbidity⁎
Miscarriages
Fetal death
n = 30
(p value)
n = 39
(p value)
n = 36
(p value)
17
(0.81)
19
(0.5)
16
(0.99)
15
(0.79)
17
(0.53)
14
(0.93)
4
(0.9)
4
(0.94)
4
(0.9)
⁎ Pregnancy morbidity was defined by APS criteria [16]. All pregnancy data percentages calculated over the total number of female who had ever been pregnant (n = 143).
M.L. Bertolaccini et al. / Thrombosis Research 130 (2012) 914–918
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Fig. 2. Correlation of aPE with aCL and anti-β2GPI. Data are given using a log-log scale. Dashed lines represent cut-off for positivity.
heterogeneity of these antibodies that increases the difficulties to attempt such a goal. These, in turns, significantly limit the clinical utility of this assay. In the context of the recent aPL meeting held in Galveston, the Non-criteria aPL task force, recognized that two further steps must be completed in order to ascertain the place of aPE in the recognition of APS, including standardization and proper validation of the aPE ELISA test along with a prospective study on a broad population with well-documented features of APS [25]. Until these objectives are achieved, there is no clear recommendation for testing for aPE in patients with SLE.
Conclusion From our data, we can conclude that aPE are frequently seen in SLE and do not correlate with other routinely tested aPL. Although more prevalent, aPE do not seem to be an independent risk factor for thrombosis or pregnancy morbidity. However, further laboratory and clinical studies are needed to define the real clinical importance of these antibodies in patients with SLE. Conflict of interest The authors declare no conflict of interest. Acknowledgements
Table 4 Multivariate analysis. p value Thrombosis Arterial thrombosis Venous thrombosis
IgG aPE IgM aPE IgG aPE IgM aPE IgG aPE IgM aPE
0.13 0.62 0.32 0.2 0.17 0.14
Variables analysed included LA, aCL, anti-β2GPI, aPT and aPS/PT, age and gender.
MLB is funded by the Louise Gergel Fellowship. This work was supported by grants from the St Thomas’ Lupus Trust. References [1] Zwaal RFA. Membrane and blood involvement in blood coagulation. Biochim Biophys Acta 1978;515:163–205. [2] Karmochkine M, Berard M, Piette JC, Cacoub P, Aillaud MF, Harlet JR, et al. Antiphosphatidylethanolamine antibodies in systemic lupus erythematosus. Lupus 1993;2:157–60.
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[3] Branch DW, Rote NS, Dostal DA, Scott JR. Association of lupus anticoagulant with antibody to phosphatidylserine. Clin Immunol Immunopathol 1987;42:63–75. [4] Falcon CR, Hoffer AM, Carreras LO. Evaluation of the clinical and laboratory associations of antiphosphatidyethanolamine. Thromb Res 1990;63:321–2. [5] Staub HL, Harris EN, Khamashta MA, Savidge G, Chahade WH, Hughes GRV. Antibody to phosphatidylethanolamine in a patient with lupus anticoagulant and thrombosis. Ann Rheum Dis 1989;48:166–9. [6] Karmochkine M, Cacoub P, Piette JC, Godeau P, Boffa MC. Antiphosphatidylethanolamine antibody as the sole antiphospholipid antibody in systemic lupus erythematosus with thrombosis. Clin Exp Rheumatol 1992;10:603–5. [7] Berard M, Chantome R, Marcelli A, Boffa MC. Antiphosphatidylethanolamine antibody as the only antiphospholipid antibodies. I. Association with thrombosis and vascular cutaneous disease. J Rheumatol 1996;23:1369–74. [8] Sanmarco M, Gayet S, Alessi MC, Audrain M, de Maistre E, Gris JC, et al. Antiphosphatidylethanolamine antibodies are associated with an increased odds ratio for thrombosis. A multicenter study with the participation of the European Forum on antiphospholipid antibodies. Thromb Haemost 2007;97:949–54. [9] Hirmerova J, Ulcova-Gallova Z, Seidlerova J, Filipovsky J, Bibkova K, Micanova Z, et al. Laboratory evaluation of antiphospholipid antibodies in patients with venous thromboembolism. Clin Appl Thromb Hemost 2010;16:318–25. [10] Sugi T, Katsunuma J, Izumi S, McIntyre JA, Makino T. Prevalence and heterogeneity of antiphosphatidylethanolamine antibodies in patients with recurrent early pregnancy losses. Fertil Steril 1999;71:1060–5. [11] Gris JC, Quere I, Sanmarco M, Boutiere B, Mercier E, Amiral J, et al. Antiphospholipid and antiprotein syndromes in non-thrombotic, non-autoimmune women with unexplained recurrent primary early foetal loss. The Nimes Obstetricians and Haematologists Study–NOHA. Thromb Haemost 2000;84:228–36. [12] Sugi T, Matsubayashi H, Inomo A, Dan L, Makino T. Antiphosphatidylethanolamine antibodies in recurrent early pregnancy loss and mid-to-late pregnancy loss. J Obstet Gynaecol Res 2004;30:326–32. [13] Bertolaccini ML, Gomez S, Pareja JF, Theodoridou A, Sanna G, Hughes GR, et al. Antiphospholipid antibody tests: spreading the net. Ann Rheum Dis 2005;64: 1639–43. [14] Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25:1271–7.
[15] Wilson WA, Gharavi AE, Koike T, Lockshin MD, Branch DW, Piette JC, et al. International consensus statement on preliminary classification criteria for definite antiphospholipid syndrome: report of an international workshop. Arthritis Rheum 1999;42:1309–11. [16] Miyakis S, Lockshin MD, Atsumi T, Branch DW, Brey RL, Cervera R, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006;4:295–306. [17] Sanmarco M. ELISA for antiphosphatidylethanolamine antibody detection: high impact of assay buffer on results. J Immunol Methods 2010;358:9–16. [18] Harris EN, Gharavi AE, Patel SP, Hughes GRV. Evaluation of the anti-cardiolipin antibody test: report of an international workshop held 4 April 1986. Clin Exp Immunol 1987;68:215–22. [19] Amengual O, Atsumi T, Khamashta MA, Koike T, Hughes GRV. Specificity of ELISA for antibody to beta 2-glycoprotein I in patients with antiphospholipid syndrome. Br J Rheumatol 1996;35:1239–43. [20] Brandt JT, Triplett DA, Alving B, Scharrer I. Criteria for the diagnosis of lupus anticoagulants: an update. On behalf of the Subcommittee on Lupus anticoagulant/ Antiphospholipid Antibody of the Scientific and Standardisation Committee of the ISTH. Thromb Haemost 1995;74:1185–90. [21] Thiagarajan P, Pengo V, Shapiro S. The use of dilute Russell viper venom time for the diagnosis of lupus anticoagulants. Blood 1986;68:869–74. [22] 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;25:1104–8. [23] Bertolaccini ML, Atsumi T, Koike T, Hughes GR, Khamashta MA. Antiprothrombin antibodies detected in two different assay systems. Prevalence and clinical significance in systemic lupus erythematosus. Thromb Haemost 2005;93:289–97. [24] Bertolaccini ML, Roch B, Amengual O, Atsumi T, Khamashta MA, Hughes GRV. Multiple antiphospholipid tests do not increase the diagnostic yield in antiphospholipid syndrome. Br J Rheumatol 1998;37:1229–32. [25] Bertolaccini ML, Amengual O, Atsumi T, Binder WL, de Laat B, Forastiero R, et al. 'Non-criteria' aPL tests: report of a task force and preconference workshop at the 13th International Congress on Antiphospholipid Antibodies, Galveston, TX, USA, April 2010, 20. Lupus; 2011. p. 191–205.
Contents lists available at SciVerse ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Regular Article
The clinical value of testing for antibodies to phosphatidylethanolamine (aPE) in patients with systemic lupus erythematosus (SLE) M.L. Bertolaccini a,⁎, V. Murru a, S. Sciascia a, G. Sanna b, M.A. Khamashta a, b a b
Lupus Research Unit, The Rayne Institute, Division of Women's Health, King's College London, UK Louise Coote Lupus Unit, Guy's and St. Thomas’ NHS Foundation Trust, St. Thomas’ Hospital, London, UK
a r t i c l e
i n f o
Article history: Received 3 May 2012 Received in revised form 30 May 2012 Accepted 6 June 2012 Available online 15 October 2012 Keywords: Antibodies to phosphatidylethanolamine Antiphospholipid syndrome Thrombosis Pregnancy morbidity Systemic lupus erythematosus
a b s t r a c t The value of testing for aPE in venous thrombosis and fetal death is in constant debate. We evaluated if testing for aPE has a diagnostic value in patients with SLE. Patients and Methods: We included 224 patients. aPE were tested by an in-house ELISA using FCS. Results: aPE were found in 41% of the patients. IgG and IgM aPE were more frequently found along with other aPL than in those negative for aPL (p = 0.003 and p = 0.01). IgG aPE were more frequently found in patients with definite APS than in those without (p = 0.003). aPE were more frequent in patients with thrombosis than in those without, particularly the IgG isotype (p = 0.03). When subdividing between venous and arterial thrombosis, only an association between IgG aPE with venous thrombosis was retained (p = 0.01). Titres of IgG aPE were significantly higher in patients with arterial or those with venous thrombosis, when compared to the patients without thrombosis (p = 0.004 and p = 0.001). Titres of IgM aPE were higher in patients with arterial thrombosis when compared to those without (p = 0.014). No associations were found between the presence of aPE and/or pregnancy morbidity. The presence of aPE did not correlate with that of any other aPL. After multivariate analysis all clinical associations failed to retain significance. Conclusions: aPE are frequently seen in SLE and do not correlate with other routinely tested aPL. Although more prevalent, aPE is not an independent risk factor for thrombosis or pregnancy morbidity in patients with SLE. © 2012 Elsevier Ltd. All rights reserved.
Introduction The antiphospholipid syndrome (APS) is a common acquired thrombophilia characterised by vascular thrombosis and/or pregnancy morbidity in the presence of antiphospholipid antibodies (aPL). aPL are a heterogeneous group of autoantibodies whose specificity is directed to phospholipid-binding proteins or their complex with phospholipids. In clinical practice, anticardiolipin antibodies (aCL), antibodies to β2 glycoprotein I (anti-β2GPI) detected by ELISA and the lupus anticoagulant (LA) detected by clotting assays are the most widely used and standardised tests for the detection of aPL. However, a variety of plasma proteins have been implicated as targets for these antibodies. Phosphatidylethanolamine (PE) is a ubiquitous phospholipid present in both the internal and external sides of normal cell membranes [1]. Antibodies to PE (aPE) have been reported to be present on a ⁎ Corresponding author at: Lupus Research Unit, The Rayne Institute, Division of Women's Health, King's College London, 4th Floor Lambeth Wing, St. Thomas’ Hospital, London SE1 7EH, UK. Tel.: + 44 2071883569; fax: + 44 2076202658. E-mail address: [email protected] (M.L. Bertolaccini). 0049-3848/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2012.06.008
range from 17% [2] to 80% [3]. In most of the cases, aPE have been reported in association with other aPL [2,4,5]. However, few reports showed aPE in the absence of aCL and/or LA [6,7]. The presence of aPE have been associated with thrombosis [5–9] and recurrent pregnancy loss [10–12] but not all studies arrived to the same conclusions [13] and, therefore, screening for aPE is still not recommended. The purpose of the present study was to evaluate if testing for aPE has a diagnostic value in patients with systemic lupus erythematosus (SLE). Patients and methods Patients This study included 224 patients, all fulfilling the 1982 criteria for SLE [14]. Of these, 59 patients fulfil criteria for definite APS [15,16] and 55 were positive for aPL without fulfilling criteria. Patients group comprised 212 women with a mean age of 42.7 ± 11.9 years and mean disease duration of 12.2 ± 8.8 years. Overall, 81 patients had a history of thrombosis (34 arterial, 27 venous and 20 both arterial and venous thrombosis). Out of 143 women who had ever been pregnant, 39 had a history of miscarriages
M.L. Bertolaccini et al. / Thrombosis Research 130 (2012) 914–918
and 36 a history of fetal death. Demographics are summarised in Table 1. Ethical approval was obtained from the Guy's and St Thomas’ Ethics committee and all patients involved in this study gave their written consent. Methods Blood collection Blood was collected by venopuncture from the antecubital vein into pre-cooled tubes containing 0.105 M sodium citrate and in non-anticoagulated tubes (Hemogard® 9NC and Hemogard® Z, respectively, Becton Dickinson, Rutherford, USA). Platelet free plasma was obtained by centrifugation at 2500 g for 20 minutes and filtration using a 0.2 μm surfactant free cellulose acetate membrane (Nalgene, Rochester, NY, USA). Plasma was stored at −80 °C until used. For sera preparation, blood was allowed to clot at room temperature and then centrifuged at 2000 g for 20 minutes. Sera were subsequently aliquoted and stored frozen at − 80 °C until use. Antibodies to phosphatidylethanolamine (aPE) testing aPE were tested as described by SanMarco et al. [17] with some modifications. Briefly, microtiter plates were coated with 50 μg/ml of phosphatidylethanolamine in methanol/chloroform (4:1) overnight at 4 °C. After washing and blocking for 1 hour with 10% fetal calf serumPBS, 50 μl of samples diluted 1:100 were added and incubated for 2 hours. After washing with PBS, alkaline phosphatase-conjugated anti-human IgG and/or IgM were added in the appropriate dilution and incubated for a further hour. All incubations were done at room temperature. After 1 hour and 3 washes with PBS, 100 μl of 1 mg/ml of p-nitrophenylphosphate (Sigma) in 1 M diethanolamine buffer (pH 9.8) were added. Optical density at 405/620 nm was measured by a Titertek Multiskan MC apparatus (Flow Laboratories, Herts, UK) and converted to units (U) with a sample showing a high binding used as a standard. The cut-off value was established on 8 AU for IgG aPE and 9 AU for IgM aPE, determined by the 99th percentile of 140 apparently healthy donors.
915
Anticoagulant-Phospholipid-dependent antibodies [20], using the Automated Coagulation Laboratory (ACL) 300R (Instrumentation Laboratory, Milan, Italy). All samples were screened using the activated partial thromboplastin time (aPTT–IL test™ APTT-SP, Instrumentation Laboratory, Italy). The dilute Russell viper venom time (dRVVT) coagulation test was performed as described by Thiagarajan et al. [21]. Antibodies to prothrombin were tested by the aPT (solid phase) and aPS/PT (phosphatidylserine-prothrombin complex) as previously reported [22,23]. Statistical analysis All statistical analysis was performed using SPSS 16.0 program (Microsoft software). Categorical analysis was expressed as odds ratio with its 95% confidence interval (OR [95%CI]), where a lower limit >1.0 was considered significant. The degree of linear association between aPE and other aPL was quantified by the Spearman's correlation method. Quantitative data were analysed by the Mann–Whitney test. All p values were determined by Fisher's exact test. A stepwise forward conditional procedure including all the variables used for the univariate analysis was used for the logistic regression analysis to explore the value of aPE. A p value of b0.05 was considered statistically significant. Results aPE were present in 92 (41%) patients. Of these, 68 had IgG aPE and 13 IgM aPE and 11 had both, IgG and IgM aPE. Prevalence of all aPL tested is shown in Table 2. IgG and IgM aPE were more frequently found in patients with aPL (n = 114) than in those without aPL (46% vs. 23%, OR 2.8 [95%CI 1.5–4.9], p = 0.003 and 15% vs. 5%, OR 3.2 [95%CI 1.2–8.5], p = 0.01, respectively). However, only IgG aPE were more frequently found in patients with definite APS than in those without (50% vs. 29%, OR 2.4 [95%CI 1.3–4.5], p = 0.003). Relationship of aPE to thrombosis and pregnancy morbidity
Other aPL The aCL ELISA was performed according to the standardised technique [18]. Anti-β2GPI were detected by ELISA as described previously [19], using irradiated microtitre plates (Nunc Maxisorp, Denmark). Plasma samples were tested for the presence of LA according to the recommended criteria from the ISTH Subcommittee on Lupus Table 1 Demographic characteristics of SLE. SLE n = 224 (%) Female (%) Mean age ± SD Mean disease duration ± SD APS* aPL (no APS) † Thrombosis‡ –Arterial thrombosis –Venous thrombosis Pregnancy morbidity§ –Miscarriages (≥1) –Miscarriages (≥3) –Fetal death
212 (95) 42.7 ± 11.9 12.2 ± 8.8 59 (26) 55 (24) 81 (36) 54 (24) 47 (20) 40 (27) 39 (27) 9 (6) 36 (25)
SLE: systemic lupus erythematosus, APS: antiphospholipid syndrome, aPL: antiphospholipid antibodies, including aCL, LA and/or anti-β2GPI. * All patients fulfilled criteria for APS [15,16]. †aPL included patients who were positive for aPL but did not fulfilled criteria (i.e. low titres and/or no clinical events attributable to APS). ‡20 patients from each group have both arterial and venous thrombosis. §Pregnancy morbidity was defined by APS criteria (ref). All pregnancy data percentages calculated over the total number of female who had ever been pregnant (n = 143).
Only IgG aPE were more frequently found in patients with thrombosis than in those without (44% vs. 30%, OR1.8 [95%CI 1.0–3.2], p = 0.03). When subdividing the thrombosis group between those with venous and those with arterial thrombosis, only the association between IgG aPE with venous thrombosis was retained (51% vs. 31%, OR2.3 [95%CI 1.2–4.4], p = 0.01). Median titres [range] of IgG aPE were significantly higher in patients with thrombosis as a whole and in those with arterial or those with venous, when compared to the patients without thrombosis (7.35 AU [1.5–81.5] and 7.23 AU [2.4–42.1] or 8.16 [1.5–81.5] vs. 5.85 AU [0.7–50.2], p = 0.004, p = 0.004 and p = 0.001 respectively). Titres of IgM aPE were higher in patients with thrombosis, particularly arterial thrombosis when compared to those without (2.0 AU [0–359.7] and 2.35 AU [0.1–359.7] vs. 1.5 AU [0–129.5], p = 0.022 and p = 0.014 respectively). All data on aPE titres is summarised in Fig. 1. No associations were found between the presence of aPE and/or pregnancy morbidity, miscarriages or fetal death in this cohort. Prevalence of aPE in women with available obstetric history is depicted in Table 3. aPE did not correlate with any other aPL including aCL and antiβ2GPI (Fig. 2) or aPT, aPS/PT and LA (data not shown). Multivariate analysis Multivariate analysis was used to examine the association of aPE to thrombosis and or pregnancy morbidity in patients with SLE after accounting for potential confounders, including other aPL, age and gender.
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Table 2 Prevalence of aPE and other aPL in SLE. aPL ⁎
SLE n = 224 (%)
aPE IgG/IgM –aPE IgG –aPE IgM aCL IgG/IgM –aCL IgG –aCL IgM Anti–β2GPI IgG/IgM –Anti-β2GPI IgG –Anti-β2GPI IgM aPT IgG/IgM –aPT IgG –aPT IgM aPS/PT IgG/IgM –aPS/PT IgG –aPS/PT IgM LA
92 79 24 126 111 46 48 38 14 68 57 15 68 55 33 56
(41) (35) (10) (56) (49) (20) (21) (16) (6) (30) (25) (6) (30) (24) (14) (25)
⁎ some patients were positive for more than 1 antibody and/or isotype. IgG/M: IgG and/or IgM. aPE: anti-phosphatidylethanolamine; aCL: anticardiolipin antibodies; anti-β2GPI: antibodies to β2 glycoprotein I, aPT: antibodies to prothrombin in solid phase; aPS/PT: antibodies to phosphatidylserine-prothrombin complex; LA: lupus anticoagulant.
After multivariate analysis, all clinical associations failed to retain significance (Table 4). Discussion In this study we attempted to analyse the value of testing for aPE as a marker of risk for thrombosis in a large cohort of patients with SLE. As aPE appear to be markers of risk for thrombosis, we evaluated the prevalence and clinical significance of aPE in a large number of patients with SLE. In this study we found an overall prevalence of aPE of 41%, with IgG and IgM isotypes present in 35% and 10% of the patients, respectively. There was a correlation between the presence of aPE and the clinical manifestations of APS and the relevance of these antibodies as markers for APS is discussed. We found that aPE are frequently found in patients with SLE, particularly in those with aPL. Although frequently seen in association with other aPL, aPE did not correlate with aCL, anti-β2GPI, aPT, aPS/ PT and/or the LA, suggesting that they are a different subpopulation of aPL. Few studies available in the literature investigated the clinical significance of aPE. The presence of aPE has been associated with thrombosis since the early 90's [5–7]. In 1998, we studied aPE in a large cohort of SLE [24]. At the time, we reported a low prevalence of aPE, now attributed to differences in the protocol used and no associations between aPE and clinical features of APS. It has recently being reported that aPE reactivity is dependent on the lipid concentration of the buffer component [17]. Whilst in our 1998 study, we used adult bovine serum (rich in lipid content), in the present study we used fetal calf serum with the consequent improvement in aPE detection. In 2007, a multicenter study followed [8] reporting IgG aPE to be an independent risk factor for venous thrombosis with an odds ratio of 6. In our present study, we found an association between the presence of aPE and the occurrence of vascular events. When patients with thrombosis were subdivided into arterial events and venous events, only IgG aPE remained associated with venous thrombosis. However, after multivariate analysis, the association between aPE and venous thrombosis failed to retain significance. In contrast, the prevalence of IgM aPE was not different between patients with arterial thrombosis and venous thrombosis.
Fig. 1. Distribution of IgG (a) and IgM (b) aPE in SLE with and without thrombosis.
aPE have been reported to be associated with pregnancy morbidity and 2 different studies have shown aPE to be a stronger independent risk factor for early miscarriages when compared to aCL and anti-β2GPI [11,12]. Interestingly, aPE have also been described as the only aPL found in most of the cases (73%). In our study, aPE were not associated to pregnancy morbidity, miscarriages or fetal death. These contradictory results can be due to the lack of consensual standardized method for the measurement of aPE and the Table 3 Prevalence of aPE in 143 women with available obstetric history. aPE
IgG/M n = 63 IgG n = 56 IgM n = 40
Pregnancy morbidity⁎
Miscarriages
Fetal death
n = 30
(p value)
n = 39
(p value)
n = 36
(p value)
17
(0.81)
19
(0.5)
16
(0.99)
15
(0.79)
17
(0.53)
14
(0.93)
4
(0.9)
4
(0.94)
4
(0.9)
⁎ Pregnancy morbidity was defined by APS criteria [16]. All pregnancy data percentages calculated over the total number of female who had ever been pregnant (n = 143).
M.L. Bertolaccini et al. / Thrombosis Research 130 (2012) 914–918
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Fig. 2. Correlation of aPE with aCL and anti-β2GPI. Data are given using a log-log scale. Dashed lines represent cut-off for positivity.
heterogeneity of these antibodies that increases the difficulties to attempt such a goal. These, in turns, significantly limit the clinical utility of this assay. In the context of the recent aPL meeting held in Galveston, the Non-criteria aPL task force, recognized that two further steps must be completed in order to ascertain the place of aPE in the recognition of APS, including standardization and proper validation of the aPE ELISA test along with a prospective study on a broad population with well-documented features of APS [25]. Until these objectives are achieved, there is no clear recommendation for testing for aPE in patients with SLE.
Conclusion From our data, we can conclude that aPE are frequently seen in SLE and do not correlate with other routinely tested aPL. Although more prevalent, aPE do not seem to be an independent risk factor for thrombosis or pregnancy morbidity. However, further laboratory and clinical studies are needed to define the real clinical importance of these antibodies in patients with SLE. Conflict of interest The authors declare no conflict of interest. Acknowledgements
Table 4 Multivariate analysis. p value Thrombosis Arterial thrombosis Venous thrombosis
IgG aPE IgM aPE IgG aPE IgM aPE IgG aPE IgM aPE
0.13 0.62 0.32 0.2 0.17 0.14
Variables analysed included LA, aCL, anti-β2GPI, aPT and aPS/PT, age and gender.
MLB is funded by the Louise Gergel Fellowship. This work was supported by grants from the St Thomas’ Lupus Trust. References [1] Zwaal RFA. Membrane and blood involvement in blood coagulation. Biochim Biophys Acta 1978;515:163–205. [2] Karmochkine M, Berard M, Piette JC, Cacoub P, Aillaud MF, Harlet JR, et al. Antiphosphatidylethanolamine antibodies in systemic lupus erythematosus. Lupus 1993;2:157–60.
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