- Email: [email protected]
Detection of antiphospholipid antibodies by automated chemiluminescence assay
Journal of Immunological Methods 379 (2012) 48–52
Contents lists available at SciVerse ScienceDirect
Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
Research paper
Detection of antiphospholipid antibodies by automated chemiluminescence assay Antonella Capozzi, Emanuela Lococo, Maria Grasso, Agostina Longo, Tina Garofalo, Roberta Misasi, Maurizio Sorice ⁎ Dipartimento di Medicina Sperimentale, “Sapienza” University, Rome, Italy
a r t i c l e
i n f o
Article history: Received 15 December 2011 Received in revised form 29 February 2012 Accepted 29 February 2012 Available online 6 March 2012 Keywords: Antiphospholipid syndrome Anticardiolipin antibodies Anti-beta2 glycoprotein I antibodies Lupus anticoagulant Thrombosis Zenit RA analyzer
a b s t r a c t The laboratory diagnosis of antiphospholipid antibody syndrome (APS) requires the demonstration of antiphospholipid antibodies (aPL) by lupus anticoagulant (LAC) measured through coagulation assays, anticardiolipin IgG or IgM antibodies (aCL) and/or anti-β2-glycoprotein I IgG or IgM antibodies (anti-β2-GPI), usually detected by ELISA. In this study we tested aCL by a new automated system using the chemiluminescence principle. Our results showed that, while almost all the sera from APS patients, positive for IgG aCL and anti-β2-GPI by ELISA, were also positive for IgG aCl by chemiluminescence, only 30.13% of patients without clinical manifestations of APS, but positive for aCL and persistently negative for anti-β2-GPI (by ELISA) and LA, confirmed the positive test by chemiluminescence. This difference was highly significant (p b 0.0001). Interestingly, this test also prompted to identify 20% of patients positive for LA, but persistently negative for both aCL and anti-β2-GPI IgG (ELISA). Thus, the new technology of automated chemiluminescence assay for measuring aPL may represent an useful tool to identify “true” APS patients. © 2012 Elsevier B.V. All rights reserved.
1. Introduction In the last few years sensitive techniques have been developed for detecting various “antiphospholipid” antibodies (aPL), among which anticardiolipin antibodies (aCL) are the best characterized. aCl were first detected in sera of patients with systemic lupus erythematosus (SLE) or related autoimmune disorders. These autoantibodies are considered responsible for the so-called antiphospholipid antibody syndrome (APS), which is characterized by arterial and/or venous thromboses and multiple abortions (Hughes, 1985; Hughes et al., 1986). Diagnosis of APS requires the combination of at least one clinical and one laboratory criterion (Wilson et al., 1999; Miyakis et al., 2006). aCL and anti-β2-
⁎ Corresponding author at: Dip. Medicina Sperimentale, “Sapienza” University of Rome, viale Regina Elena 324, 00161 Rome, Italy. Tel.: + 39 6 49972675; fax: + 39 6 4456229. E-mail address: [email protected] (M. Sorice). 0022-1759/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2012.02.020
glycoprotein-I (anti-β2-GPI) antibodies, detected by enzyme linked immunosorbent assay (ELISA) and the lupus anticoagulant (LA), detected by clotting assays, are the recommended tests for the detection of aPL (Tincani et al., 1998; Bertolaccini et al., 2005). Indeed, aPL represent a heterogeneous family of antibodies that react with serum phospholipid-binding plasma proteins, among which β2-GPI represents the main protein cofactor (Galli et al., 1993; De Laat et al., 2004). In addition, protein S (Sorice et al., 1994a, 1996), protein C (Oosting et al., 1993), prothrombin (Arvieux et al., 1995; Sorice et al., 1998), annexin V (Kaburaki et al., 1997), annexin II (Salle et al., 2008) or vimentin (Ortona et al., 2010) have been also demonstrated as antigenic targets for these autoantibodies. “Pure” aPL can be detected by thin layer chromatography (TLC) immunostaining (Sorice et al., 1994b). Subsequent studies revealed that aCL may occur in a wide range of other conditions, including mainly infectious diseases, but also neurological disorders, treatment with particular drugs or in apparently healthy individuals (Devreese and
A. Capozzi et al. / Journal of Immunological Methods 379 (2012) 48–52
Hoylaerts, 2009). However, only autoimmune-type aCL are usually dependent on the presence of β2-GPI (McNeil et al., 1990; Gibril and Jensen, 1997) and, possibly, other plasma proteins, such as prothrombin and annexin V (Triplett, 1995; Arvieux et al., 1995). Despite the guidelines for measuring aCL proposed by the standardization group of the European Forum on Antiphospholipid antibodies (Tincani et al., 2004, 2009; Reber et al., 2004), the laboratory diagnosis of the APS remains a challenge for each laboratory worker in the field. Automatization can improve the reproducibility and reduce interlaboratory variation. Recently, a new chemiluminescence automated system has been proposed (Persijn et al., 2011). The new technology of chemiluminescence for measuring aPL showed good performance characteristics. Interpretation of results with a cut-off value associated with a good discrimination for disease, resulted in a good sensitivity and specificity. The aim of this study was to evaluate the diagnostic value of aPL, measured by this new chemiluminescence automated system. Our results revealed that the presence of aCL was significantly higher in APS patients persistently positive for LA, aCL and anti-β2-GPI, as compared to those positive for aCL in ELISA, negative for anti-β2-GPI and LA, without clinical manifestations of APS. 2. Materials and methods
49
163 subjects (35 males, 128 females, mean age 40.5, range 22–75 years) were healthy blood donors, negative for anti-β2-GPI and LA. In this group, none of the subjects showed signs or symptoms of APS. All serum samples were obtained from Department of Experimental Medicine, “Sapienza” University of Rome. 2.3. Autoantibody assay IgG and IgM aCL were detected by ELISA, using QUANTA Lite™ detection kit (INOVA Diagnostic Inc., San Diego, CA, USA) assay used as an internal reference a representative standard curve, using IgG Sapporo HCAL monoclonal antibody or IgM EY2C9 monoclonal antibody (Ichikawa et al., 1999). This material was from laboratory of Dr Takao Koike. Results are expressed in GPL or MPL. Sera > 15 GPL or MPL U/ml (U = units) were considered as positive. All the positive sera were confirmed by ELISA detection system (ORGENTEC Diagnostika GmbH, Mainz, Germany). IgG and IgM anti-β2-GPI were detected by ELISA, using QUANTA Lite™ detection kit (INOVA Diagnostic Inc.). This assay used as an internal reference a representative standard curve, using IgG or IgM human antibody. Sera > 20 Standard Units of IgG (SGU) or IgM (SMU) anti-β2-GPI were considered as positive.
2.1. Samples preparation 2.4. Lupus anticoagulant assay After having obtained an informed consent from each patient, a venous bleeding was performed. Sera were prepared by centrifugation at 3000g for 10 minutes at room temperature. For platelet-poor plasma preparation, blood was drawn in 3.2% sodium citrate tubes (one part sodium citrate to nine parts venous blood) and then centrifuged at 2500g for 20 minutes at room temperature. All samples were kept at − 20 °C for further analysis. 2.2. Patients This study included 314 consecutive subjects: 63 patients (13 males, 50 females, mean age 43.3 years, range 4–77 years) were affected by APS, diagnosed according to the classification criteria for definite APS (Miyakis et al., 2006), persistently positive for LA, aCL and anti-β2-GPI, as determined by ELISA. In this group, 15 patients were affected by primary antiphospholipid syndrome (PAPS) and 48 by secondary antiphospholipid syndrome (SAPS). All the patients had shown venous and/or arterial thrombosis. 73 patients (25 males, 58 females, mean age 38.9 years, range 3–71 years) did not show clinical manifestations of APS, but were positive for aCL and persistently negative for anti-β2-GPI (by ELISA) and LA. In this group, 16 were suffering from infectious diseases, 30 from rheumatic disorders, 3 from thrombocytopenia, 1 from pregnancy disorders, 3 from neurological disorders, 5 from gastrointestinal disorders, 2 ocular disorders and 13 from other APS-unrelated disorders. 15 patients (4 males, 11 females, mean age 39.9 years, range 4–76 years) were LA-positive, but negative in ELISA for aCL and anti-β2-GPI. In this group, all the patients had shown venous and/or arterial thrombosis.
LA was studied in two coagulation systems, a dilute sensitized activated partial thromboplastin time (aPTT) and a dilute Russell's viper venom time (dRVVT), followed by a confirmation test, using reagents and instrumentation by Hemoliance Instrumentation Laboratory (Lexington, MA, USA). 2.5. Chemiluminescence assay IgG and IgM aCL were also tested by chemiluminescence assay by Zenit RA immunoanalyzer (A. Menarini Diagnostics, Florence, Italy) (Persijn et al., 2011). Zenit RA, a random-access immunoanalyzer, uses a two-step immunoassay method based on the principle of chemiluminescence. CL/β2-GPI complex is used to coat magnetic particles (solid phase) and a human anti-IgG or antiIgM is labeled with an acridine ester derivative (conjugate). The source of CL is bovine heart. During the first incubation, the specific antibodies present in the sample, in the calibrators, or in the controls bind with the solid phase. During the second incubation, the conjugate reacts with the antibodies captured on the solid phase. After each incubation, the material that has not bonded with the solid phase is removed by suction and repeated washing. The quantity of marked conjugate bonded to the solid phase is evaluated by chemiluminescent reaction and measurement of the light signal. The generated signal, measured in RLU (Relative Light Units), is indicative of the concentration of the specific antibodies present in the sample, in the calibrators, and in the controls. For aCL IgG or IgM the concentrations of the calibrators are expressed in GPL or MPL U/ml (U= units) and calibrated against the “Harris” reference sera. Every sample was analyzed
50
A. Capozzi et al. / Journal of Immunological Methods 379 (2012) 48–52
Table 1 Results of anti-CL in chemiluminescence. Patients selected by ELISA
Anti-CL + Anti-β2-GPI + (N = 63)
Results in chemiluminescence
IgG 55/63* 87.30%
Anti-CL + Anti-β2-GPI(N = 73) IgM 17/63^ 26.98%
IgG 22/73* 30.13%
Anti-CLLA+ (N = 15) IgM 9/73^ 12.32%
IgG 3/15 20%
Anti-CLAnti-β2-GPI(N = 163) IgM 3/15 20%
IgG 0/163 0%
IgM 3/163 1.84%
anti-CL, anticardiolipin antibodies; anti-β2-GPI, anti-beta2-glycoprotein I antibodies; LA, lupus anticoagulant. * p b 0.0001. ^ p = 0.03.
in duplicate (calibrators, controls, reference population and patient samples). Sera > 20 GPL or 10 MPL were considered as positive, according to manufacturer's indication. Quality control material, provided by the manufacturer, was analyzed in every run. The APS IgM or IgG control set provides a ready-to-use positive control, containing a known quantity of aCL antibodies and a negative control containing normal human serum. 2.6. Statistical analysis All the statistical procedures were performed by SAS/STAT Software provided by SAS Institute Inc. (Cary, North Carolina, USA). Sensitivity, specificity, positive and negative predictive values for chemiluminescence assay were calculated against the clinical background of the patients. For comparison of categorical variables or percentages Chi square test (χ 2) was employed. p Values less than 0.05 were considered as significant. 3. Results In this study a new automated system applying chemiluminescence technology for aPL detection was employed. Since there is no gold standard for aCL antibody assay, we preliminary calculated in-house cut-off values (Tincani et al., 2004), using the 99th percentile (Miyakis et al., 2006) of a normal population of 163 healthy blood donors. Calculated cut-offs by the 99th percentile for IgG and IgM aCL were very low (2 GPL/ml and 6 MPL/ml, respectively). Thus, according to Persijn et al. (2011), we assumed for IgG and IgM aCL the cut-off values indicated by the manufacturer (20 GPL and 10 MPL, respectively). Assuming these cut-off values, our analysis revealed that 55/63 (87.3%) patients affected by APS, persistently positive
for LA, aCL and anti-β2-GPI (by ELISA), were positive for IgG aCL and 17/63 (26.98%) for IgM aCL by chemiluminescence (Table 1). Interestingly, only 30.13% of sera from patients without clinical manifestations of APS, but positive for aCL (ELISA) and persistently negative for anti-β2-GPI (ELISA) and LA were also positive for IgG aCL by chemiluminescence. This difference was highly significant (p b 0.0001). Of note, in 20% of APS patients who were LA-positive, but negative in ELISA for aCL and anti-β2-GPI, we detected aCL by chemiluminescence (Table 1). It indicates that this method may be useful in same cases in which the “classical” aPL ELISA tests are negative. None of the sera from healthy blood donors displayed aCL antibodies by chemiluminescence, 5/163 (3.06%) IgG aCL by ELISA and 0/163 IgM aCL by ELISA. The analysis of sensitivity and specificity revealed that IgG test by automated chemiluminescence is less sensitive, but more specific than ELISA (Table S1). When APS sera under test were grouped according to the GPL values obtained in ELISA tests, our results revealed that the number of positive sera by chemiluminescence was higher in the group >100 GPL (19/20, 95%), as compared to that between 50 and 100 GPL (20/23, 86.9%) and that between 15 and 50 GPL (17/21, 80.9%) (Table 2). When APS sera under test were grouped according to the clinical features (Table 3) we observed that the occurrence of IgG aCL by chemiluminescence was virtually the same between patients with PAPS and patients with SAPS (p > 0.05), whereas the number of positive sera for IgM by chemiluminescence was higher in the group of PAPS as compared to SAPS patients (p b 0.001). No correlation between aCL and APS activity has been demonstrated. As expected, in the group of patients without clinical manifestations of APS, but positive for aCL (ELISA) and persistently negative for anti-β2-GPI (ELISA) and LA, the highest positivity of aCL by chemiluminescence was detected among patients
Table 2 Results of anti-CL in chemiluminescence in APS patients grouped according to the GPL values. Patients selected by ELISA
APS patients 15–50 GPL (N = 21)
Results in chemiluminescence
IgG 17/21§ 80.95%
APS patients > 100 GPL (N = 20)
APS patients 50–100 GPL (N = 23) IgM 7/21# 33.33%
IgG 20/23* 86.95%
anti-CL, anticardiolipin antibodies; APS patients, patients with antiphospholipid antibody syndrome; * p > 0.05. ^ p > 0.05. § p > 0.05. # p > 0.05.
IgM 6/23^ 26.08%
IgG 18/19*§ 94.73%
IgM 4/19^# 21.05%
A. Capozzi et al. / Journal of Immunological Methods 379 (2012) 48–52 Table 3 Results of anti-CL in chemiluminescence in APS patients. APS patients (N = 63)
PAPS (N = 15)
SAPS (N = 48)
Results in chemiluminescence
IgG 13/15* 86.66%
IgG 43/48* 89.58%
IgM 9/15^ 60%
IgM 8/48^ 16.66%
anti-CL, anticardiolipin antibodies; PAPS, patients with primary antiphospholipid antibody syndrome; SAPS, patients with secondary antiphospholipid antibody syndrome; * p > 0.05. ^ p b 0.0001.
with rheumatic disorders (50%), followed by patients with neurological disorders (33.3%) or infectious diseases (25%). All these single groups revealed a significantly lower occurrence of aCL by chemiluminescence as compared to APS patients (p b 0.0001) (Table 4). 4. Discussion In this study we tested aCL by a new automated system, using the chemiluminescence principle. Several methods have been employed for detection of aPl. However, “classical” aCL antibodies are usually present not only in APS patients, but also in patients with different autoimmune or infectious diseases (Sorice et al., 2000). Moreover, aCL have limitations according to robustness, reproducibility, standardization and clinical relevance (Devreese and Hoylaerts, 2009). On the other hand, anti-β2-GPI, as well as LA, are highly specific for APS patients, but not very sensitive. Thus, in this study, we decided to test for β2-GPI-dependent aCL selected sera obtained from two main groups of patients, by a new automated system using the chemiluminescence principle. Our results revealed a good agreement between automated chemiluminescence and ELISA in the APS patient population, according to previous data by Persijn et al. (2011). In fact, almost all the sera from APS patients, positive for IgG aCL and anti-β2-GPI, by ELISA, were also positive for IgG aCl by automated chemiluminescence. On the contrary, only 30.13% of patients without clinical manifestations of APS, but positive for aCL and negative for anti-β2-GPI (by ELISA) and LA, confirmed the positive test by chemiluminescence. Thus, automated chemiluminescence, although less sensitive than ELISA, appears to be more useful for identifying APS patients. This difference was not very surprising,
Table 4 Results of anti-CL in chemiluminescence in patients, positive for anti-CL and negative for anti-β2-GPI grouped according to the clinical features. Patients
No
No of aCL positive in chemiluminescence
%
Infectious diseases Arthritis Thrombocitopenia Pregnancy disorders Neurological disorders Gastrointestinal disorders Ocular disorders Other APS unrelated disorders
16 30 3 1 3 5 2 13
4 15 0 0 1 0 0 2
25% 50% 0% 0% 33.33% 0% 0% 15.38%
51
since this new automated system does differ from ELISA not only for the detection system, but also for antigen coating. Indeed, in our automated system a CL/β2-GPI complex was used to coat magnetic particles. Since it is well known that the antigen coating is a key element for aCL detection (Capuano et al., 2007), it is likely that the discrepancies between automated chemiluminescence and ELISA in detecting antibodies against CL may be due to the different antigenic presentation of phospholipid/β2-GPI complex on magnetic particles compared to the surface of microtitre wells. Using this method, the occurrence of aCL was significantly higher in APS patients, as compared to patients without clinical manifestations of APS, but positive for aCL by ELISA. This finding suggests that this test is able to specifically detect aPl in those patients who fulfilled the clinical criteria of APS. Interestingly, this test also prompted to identify 20% of patients positive for LA, but persistently negative for both aCL and anti-β2-GPI IgG (ELISA). This finding supports the view that aCL detection by chemiluminescence may be useful to reveal antibodies with a pathogenic role in APS. In the last few years it was demonstrated that antibodies responsible for the reaction in the aCL test also bind other negatively charged phospholipids, as well as β2-glycoprotein I (Galli et al., 1993; De Laat et al., 2004), prothrombin (Arvieux et al., 1995), protein S (Sorice et al., 1994a, 1996), protein C (Oosting et al., 1993), annexin V (Kaburaki et al., 1997), annexin II (Salle et al., 2008) and vimentin (Ortona et al., 2010). However, it is generally accepted that the combination of LAC and aCL have good correlation with the diagnosis of APS (Shoenfeld et al., 2008) and that at present routine testing of other aPL is expensive and does not significantly improve diagnosis. Taken together, our results suggest that the new technology of automated chemiluminescence assay for measuring aPL may represent a useful tool to identify “true” APS patients. References Arvieux, J., Darnige, L., Caron, C., Reber, G., Bensa, J.C., Colomb, M.G., 1995. Development of an ELISA for autoantibodies to prothrombin showing their prevalence in patients with lupus anticoagulants. Thromb. Haemost. 74, 1120. Bertolaccini, M.L., Gomez, S., Pareja, J.F., Theodoridou, A., Sanna, G., Hughes, G.R.V., Khamashta, M.A., 2005. Antiphospholipid antibody tests: spreading the net. Ann. Rheum. Dis. 64, 1639. Capuano, F., Grasso, V., Belforte, L., Pallavicini, L., Tincani, A., Andreoli, L., Bonelli, F., 2007. Development of automated assays for anticardiolipin antibodies determination: addressing antigen and standardization issues. Ann. N. Y. Acad. Sci. 1109, 493. De Laat, H., Derksen, R., De Groot, P.G., 2004. β2-glycoprotein I, the playmaker of the antiphospholipid syndrome. Clin. Immunol. 112, 161. Devreese, K., Hoylaerts, M.F., 2009. Laboratory diagnosis of the antiphospholipid syndrome: a plethora of obstacles to overcome. Eur. J. Haematol. 83, 1. Galli, M., Barbui, T., Zwaal, R.F.A., Confurius, P., Brevers, E.M., 1993. Antiphospholipid antibodies: involvement of protein cofactors. Haematology 78, 1. Gibril, F., Jensen, R.T., 1997. Comparative analysis of diagnostic techniques for localization of gastrointestinal neuroendocrine tumors. Yale J. Biol. Med. 70, 509. Hughes, G.R.V., 1985. The anticardiolipin syndrome. Clin. Exp. Rheumatol. 3, 285. Hughes, G.R.V., Harris, E.N., Gharavi, A.E., 1986. The anticardiolipin syndrome. J. Rheumatol. 13, 486. Ichikawa, K., Tsutsumi, A., Atsumi, T., Matsuura, E., Kobayashi, S., Hughes, G.R.V., Khamashta, M.A., Koike, T., 1999. A chimeric antibody with the human gamma1 constant region as a putative standard for assays to detect IgG beta2-glycoprotein I-dependent anticardiolipin and anti-beta2glycoprotein I antibodies. Arthritis Rheum. 42, 2461.
52
A. Capozzi et al. / Journal of Immunological Methods 379 (2012) 48–52
Kaburaki, J., Kuwana, M., Yamamoto, M., Kawai, S., Ikeda, Y., 1997. Clinical significance of anti-annexin V antibodies in patients with systemic lupus erythematosus. Am. J. Hematol. 54, 209. McNeil, H.P., Simpson, R.J., Chesterman, C.N., Krilis, S.A., 1990. Anti-phospholipid antibodies are directed against a complex antigen that includes a lipidbinding inhibitor of coagulation: beta 2-glycoprotein I (apolipoprotein H). Proc. Natl. Acad. Sci. U. S. A. 87, 4120. Miyakis, S., Lockshin, M.D., Atsumi, T., Branch, D.W., Brey, R.L., Cervera, R., Derksen, R.H., De Groot, P.G., Koike, T., Meroni, P.L., Reber, G., Shoenfeld, Y., Tincani, A., Vlachoyiannopoulos, P.G., Krilis, S.A., 2006. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J. Thromb. Haemost. 4, 295. Oosting, J.D., Derksen, R.H., Bobbink, I.W., Hackeng, T.M., Bouma, B.N., De Groot, P.G., 1993. Antiphospholipid antibodies directed against a combination of phospholipids with prothrombin, protein C, or protein S: an explanation for their pathogenic mechanism? Blood 81, 2618. Ortona, E., Capozzi, A., Colasanti, T., Conti, F., Alessandri, C., Longo, A., Garofalo, T., Margutti, P., Misasi, R., Khamashta, M.A., Hughes, G.R.V., Valesini, G., Sorice, M., 2010. Vimentin/cardiolipin complex as a new antigenic target of the Antiphospholipid Syndrome. Blood 116, 2960. Persijn, L., Decavele, A.S., Schouwers, S., Devreese, K., 2011. Evaluation of a new set of automated chemiluminescense assays for anticardiolipin and anti-beta2-glycoprotein I antibodies in the laboratory diagnosis of the antiphospholipid syndrome. Thromb. Res. 128, 565. Reber, G., Tincani, A., Sanmarco, M., De Moerloose, P., Boffa, M.C., 2004. Proposals for the measurement of anti-β2-glycoprotein I antibodies. Standardization Group of the European Forum on Antiphospholipid Antibodies. J. Thromb. Haemost. 2, 1860. Salle, V., Mazière, J.C., Smail, A., Cévallos, R., Mazière, C., Fuentes, V., Tramier, B., Makdassi, R., Choukroun, G., Vittecoq, O., Goëb, V., Ducroix, J.P., 2008. Anti-annexin II antibodies in systemic autoimmune diseases and antiphospholipid syndrome. J. Clin. Immunol. 28, 291. Shoenfeld, Y., Twig, G., Katz, U., Sherer, Y., 2008. Autoantibody explosion in antiphospholipid syndrome. J. Autoimmun. 30, 74.
Sorice, M., Griggi, T., Circella, A., Garofalo, T., D'Agostino, F., Pittoni, V., Pontieri, G.M., Lenti, L., Valesini, G., 1994a. Detection of antiphospholipid antibodies by immunostaining on thin layer chromatography plates. J. Immunol. Methods 173, 49. Sorice, M., Griggi, T., Circella, A., Lenti, L., Arcieri, P., Di Nucci, G.D., Mariani, G., 1994b. Protein S antibodies in acquired protein S deficiencies. Blood 83, 2383. Sorice, M., Arcieri, P., Griggi, T., Circella, A., Misasi, R., Lenti, L., Di Nucci, G.D., Mariani, G., 1996. Inhibition of protein S by autoantibodies in patients with acquired protein S deficiency. Thromb. Haemost. 75, 555. Sorice, M., Pittoni, V., Circella, A., Misasi, R., Conti, F., Longo, A., Pontieri, G.M., Valesini, G., 1998. Anti-prothrombin but not “pure” anti-cardiolipin antibodies are associated with the clinical features of the antiphospholipid antibody syndrome. Thromb. Haemost. 80, 713. Sorice, M., Pittoni, V., Griggi, T., Losardo, A., Leri, O., Magno, M.S., Misasi, R., Valesini, G., 2000. Specificity of antiphospholipid antibodies in infectious mononucleosis: a role for anti-cofactor protein antibodies. Clin. Exp. Immunol. 120, 301. Tincani, A., Balestrieri, G., Spatola, L., Cinquini, M., Meroni, P.L., Roubey, R.A., 1998. Anticardiolipin and anti-beta2 glycoprotein immunoassays in the diagnosis of antiphospholipid syndrome. Clin. Exp. Rheumatol. 16, 396. Tincani, A., Allegri, F., Balestrieri, G., Reber, G., Sanmarco, M., Meroni, P., Boffa, M.C., 2004. Minimal requirementsfor antiphospholipid antibodies ELISAs proposed by the European forum on the antiphospholipid antibodies. Thromb. Res. 114, 553. Tincani, A., Filippini, M., Scarsi, M., Galli, M., Meroni, P.L., 2009. European attempts for the standardisation of the antiphospholipid antibodies. Lupus 18, 913. Triplett, D.A., 1995. Protean clinical presentation of antiphospholipid– protein antibodies (APA). Thromb. Haemost. 74, 329. Wilson, W.A., Gharavi, A.E., Koike, T., Lockshin, M.D., Branch, D.W., Piette, J.C., Brey, R., Derksen, R., Harris, E.N., Hughes, G.R.V., Triplett, D.A., Khamashta, M.A., 1999. International consensus statement on preliminary classification criteria for definite antiphospholipid syndrome. Arthritis Rheum. 42, 1309.
Contents lists available at SciVerse ScienceDirect
Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
Research paper
Detection of antiphospholipid antibodies by automated chemiluminescence assay Antonella Capozzi, Emanuela Lococo, Maria Grasso, Agostina Longo, Tina Garofalo, Roberta Misasi, Maurizio Sorice ⁎ Dipartimento di Medicina Sperimentale, “Sapienza” University, Rome, Italy
a r t i c l e
i n f o
Article history: Received 15 December 2011 Received in revised form 29 February 2012 Accepted 29 February 2012 Available online 6 March 2012 Keywords: Antiphospholipid syndrome Anticardiolipin antibodies Anti-beta2 glycoprotein I antibodies Lupus anticoagulant Thrombosis Zenit RA analyzer
a b s t r a c t The laboratory diagnosis of antiphospholipid antibody syndrome (APS) requires the demonstration of antiphospholipid antibodies (aPL) by lupus anticoagulant (LAC) measured through coagulation assays, anticardiolipin IgG or IgM antibodies (aCL) and/or anti-β2-glycoprotein I IgG or IgM antibodies (anti-β2-GPI), usually detected by ELISA. In this study we tested aCL by a new automated system using the chemiluminescence principle. Our results showed that, while almost all the sera from APS patients, positive for IgG aCL and anti-β2-GPI by ELISA, were also positive for IgG aCl by chemiluminescence, only 30.13% of patients without clinical manifestations of APS, but positive for aCL and persistently negative for anti-β2-GPI (by ELISA) and LA, confirmed the positive test by chemiluminescence. This difference was highly significant (p b 0.0001). Interestingly, this test also prompted to identify 20% of patients positive for LA, but persistently negative for both aCL and anti-β2-GPI IgG (ELISA). Thus, the new technology of automated chemiluminescence assay for measuring aPL may represent an useful tool to identify “true” APS patients. © 2012 Elsevier B.V. All rights reserved.
1. Introduction In the last few years sensitive techniques have been developed for detecting various “antiphospholipid” antibodies (aPL), among which anticardiolipin antibodies (aCL) are the best characterized. aCl were first detected in sera of patients with systemic lupus erythematosus (SLE) or related autoimmune disorders. These autoantibodies are considered responsible for the so-called antiphospholipid antibody syndrome (APS), which is characterized by arterial and/or venous thromboses and multiple abortions (Hughes, 1985; Hughes et al., 1986). Diagnosis of APS requires the combination of at least one clinical and one laboratory criterion (Wilson et al., 1999; Miyakis et al., 2006). aCL and anti-β2-
⁎ Corresponding author at: Dip. Medicina Sperimentale, “Sapienza” University of Rome, viale Regina Elena 324, 00161 Rome, Italy. Tel.: + 39 6 49972675; fax: + 39 6 4456229. E-mail address: [email protected] (M. Sorice). 0022-1759/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2012.02.020
glycoprotein-I (anti-β2-GPI) antibodies, detected by enzyme linked immunosorbent assay (ELISA) and the lupus anticoagulant (LA), detected by clotting assays, are the recommended tests for the detection of aPL (Tincani et al., 1998; Bertolaccini et al., 2005). Indeed, aPL represent a heterogeneous family of antibodies that react with serum phospholipid-binding plasma proteins, among which β2-GPI represents the main protein cofactor (Galli et al., 1993; De Laat et al., 2004). In addition, protein S (Sorice et al., 1994a, 1996), protein C (Oosting et al., 1993), prothrombin (Arvieux et al., 1995; Sorice et al., 1998), annexin V (Kaburaki et al., 1997), annexin II (Salle et al., 2008) or vimentin (Ortona et al., 2010) have been also demonstrated as antigenic targets for these autoantibodies. “Pure” aPL can be detected by thin layer chromatography (TLC) immunostaining (Sorice et al., 1994b). Subsequent studies revealed that aCL may occur in a wide range of other conditions, including mainly infectious diseases, but also neurological disorders, treatment with particular drugs or in apparently healthy individuals (Devreese and
A. Capozzi et al. / Journal of Immunological Methods 379 (2012) 48–52
Hoylaerts, 2009). However, only autoimmune-type aCL are usually dependent on the presence of β2-GPI (McNeil et al., 1990; Gibril and Jensen, 1997) and, possibly, other plasma proteins, such as prothrombin and annexin V (Triplett, 1995; Arvieux et al., 1995). Despite the guidelines for measuring aCL proposed by the standardization group of the European Forum on Antiphospholipid antibodies (Tincani et al., 2004, 2009; Reber et al., 2004), the laboratory diagnosis of the APS remains a challenge for each laboratory worker in the field. Automatization can improve the reproducibility and reduce interlaboratory variation. Recently, a new chemiluminescence automated system has been proposed (Persijn et al., 2011). The new technology of chemiluminescence for measuring aPL showed good performance characteristics. Interpretation of results with a cut-off value associated with a good discrimination for disease, resulted in a good sensitivity and specificity. The aim of this study was to evaluate the diagnostic value of aPL, measured by this new chemiluminescence automated system. Our results revealed that the presence of aCL was significantly higher in APS patients persistently positive for LA, aCL and anti-β2-GPI, as compared to those positive for aCL in ELISA, negative for anti-β2-GPI and LA, without clinical manifestations of APS. 2. Materials and methods
49
163 subjects (35 males, 128 females, mean age 40.5, range 22–75 years) were healthy blood donors, negative for anti-β2-GPI and LA. In this group, none of the subjects showed signs or symptoms of APS. All serum samples were obtained from Department of Experimental Medicine, “Sapienza” University of Rome. 2.3. Autoantibody assay IgG and IgM aCL were detected by ELISA, using QUANTA Lite™ detection kit (INOVA Diagnostic Inc., San Diego, CA, USA) assay used as an internal reference a representative standard curve, using IgG Sapporo HCAL monoclonal antibody or IgM EY2C9 monoclonal antibody (Ichikawa et al., 1999). This material was from laboratory of Dr Takao Koike. Results are expressed in GPL or MPL. Sera > 15 GPL or MPL U/ml (U = units) were considered as positive. All the positive sera were confirmed by ELISA detection system (ORGENTEC Diagnostika GmbH, Mainz, Germany). IgG and IgM anti-β2-GPI were detected by ELISA, using QUANTA Lite™ detection kit (INOVA Diagnostic Inc.). This assay used as an internal reference a representative standard curve, using IgG or IgM human antibody. Sera > 20 Standard Units of IgG (SGU) or IgM (SMU) anti-β2-GPI were considered as positive.
2.1. Samples preparation 2.4. Lupus anticoagulant assay After having obtained an informed consent from each patient, a venous bleeding was performed. Sera were prepared by centrifugation at 3000g for 10 minutes at room temperature. For platelet-poor plasma preparation, blood was drawn in 3.2% sodium citrate tubes (one part sodium citrate to nine parts venous blood) and then centrifuged at 2500g for 20 minutes at room temperature. All samples were kept at − 20 °C for further analysis. 2.2. Patients This study included 314 consecutive subjects: 63 patients (13 males, 50 females, mean age 43.3 years, range 4–77 years) were affected by APS, diagnosed according to the classification criteria for definite APS (Miyakis et al., 2006), persistently positive for LA, aCL and anti-β2-GPI, as determined by ELISA. In this group, 15 patients were affected by primary antiphospholipid syndrome (PAPS) and 48 by secondary antiphospholipid syndrome (SAPS). All the patients had shown venous and/or arterial thrombosis. 73 patients (25 males, 58 females, mean age 38.9 years, range 3–71 years) did not show clinical manifestations of APS, but were positive for aCL and persistently negative for anti-β2-GPI (by ELISA) and LA. In this group, 16 were suffering from infectious diseases, 30 from rheumatic disorders, 3 from thrombocytopenia, 1 from pregnancy disorders, 3 from neurological disorders, 5 from gastrointestinal disorders, 2 ocular disorders and 13 from other APS-unrelated disorders. 15 patients (4 males, 11 females, mean age 39.9 years, range 4–76 years) were LA-positive, but negative in ELISA for aCL and anti-β2-GPI. In this group, all the patients had shown venous and/or arterial thrombosis.
LA was studied in two coagulation systems, a dilute sensitized activated partial thromboplastin time (aPTT) and a dilute Russell's viper venom time (dRVVT), followed by a confirmation test, using reagents and instrumentation by Hemoliance Instrumentation Laboratory (Lexington, MA, USA). 2.5. Chemiluminescence assay IgG and IgM aCL were also tested by chemiluminescence assay by Zenit RA immunoanalyzer (A. Menarini Diagnostics, Florence, Italy) (Persijn et al., 2011). Zenit RA, a random-access immunoanalyzer, uses a two-step immunoassay method based on the principle of chemiluminescence. CL/β2-GPI complex is used to coat magnetic particles (solid phase) and a human anti-IgG or antiIgM is labeled with an acridine ester derivative (conjugate). The source of CL is bovine heart. During the first incubation, the specific antibodies present in the sample, in the calibrators, or in the controls bind with the solid phase. During the second incubation, the conjugate reacts with the antibodies captured on the solid phase. After each incubation, the material that has not bonded with the solid phase is removed by suction and repeated washing. The quantity of marked conjugate bonded to the solid phase is evaluated by chemiluminescent reaction and measurement of the light signal. The generated signal, measured in RLU (Relative Light Units), is indicative of the concentration of the specific antibodies present in the sample, in the calibrators, and in the controls. For aCL IgG or IgM the concentrations of the calibrators are expressed in GPL or MPL U/ml (U= units) and calibrated against the “Harris” reference sera. Every sample was analyzed
50
A. Capozzi et al. / Journal of Immunological Methods 379 (2012) 48–52
Table 1 Results of anti-CL in chemiluminescence. Patients selected by ELISA
Anti-CL + Anti-β2-GPI + (N = 63)
Results in chemiluminescence
IgG 55/63* 87.30%
Anti-CL + Anti-β2-GPI(N = 73) IgM 17/63^ 26.98%
IgG 22/73* 30.13%
Anti-CLLA+ (N = 15) IgM 9/73^ 12.32%
IgG 3/15 20%
Anti-CLAnti-β2-GPI(N = 163) IgM 3/15 20%
IgG 0/163 0%
IgM 3/163 1.84%
anti-CL, anticardiolipin antibodies; anti-β2-GPI, anti-beta2-glycoprotein I antibodies; LA, lupus anticoagulant. * p b 0.0001. ^ p = 0.03.
in duplicate (calibrators, controls, reference population and patient samples). Sera > 20 GPL or 10 MPL were considered as positive, according to manufacturer's indication. Quality control material, provided by the manufacturer, was analyzed in every run. The APS IgM or IgG control set provides a ready-to-use positive control, containing a known quantity of aCL antibodies and a negative control containing normal human serum. 2.6. Statistical analysis All the statistical procedures were performed by SAS/STAT Software provided by SAS Institute Inc. (Cary, North Carolina, USA). Sensitivity, specificity, positive and negative predictive values for chemiluminescence assay were calculated against the clinical background of the patients. For comparison of categorical variables or percentages Chi square test (χ 2) was employed. p Values less than 0.05 were considered as significant. 3. Results In this study a new automated system applying chemiluminescence technology for aPL detection was employed. Since there is no gold standard for aCL antibody assay, we preliminary calculated in-house cut-off values (Tincani et al., 2004), using the 99th percentile (Miyakis et al., 2006) of a normal population of 163 healthy blood donors. Calculated cut-offs by the 99th percentile for IgG and IgM aCL were very low (2 GPL/ml and 6 MPL/ml, respectively). Thus, according to Persijn et al. (2011), we assumed for IgG and IgM aCL the cut-off values indicated by the manufacturer (20 GPL and 10 MPL, respectively). Assuming these cut-off values, our analysis revealed that 55/63 (87.3%) patients affected by APS, persistently positive
for LA, aCL and anti-β2-GPI (by ELISA), were positive for IgG aCL and 17/63 (26.98%) for IgM aCL by chemiluminescence (Table 1). Interestingly, only 30.13% of sera from patients without clinical manifestations of APS, but positive for aCL (ELISA) and persistently negative for anti-β2-GPI (ELISA) and LA were also positive for IgG aCL by chemiluminescence. This difference was highly significant (p b 0.0001). Of note, in 20% of APS patients who were LA-positive, but negative in ELISA for aCL and anti-β2-GPI, we detected aCL by chemiluminescence (Table 1). It indicates that this method may be useful in same cases in which the “classical” aPL ELISA tests are negative. None of the sera from healthy blood donors displayed aCL antibodies by chemiluminescence, 5/163 (3.06%) IgG aCL by ELISA and 0/163 IgM aCL by ELISA. The analysis of sensitivity and specificity revealed that IgG test by automated chemiluminescence is less sensitive, but more specific than ELISA (Table S1). When APS sera under test were grouped according to the GPL values obtained in ELISA tests, our results revealed that the number of positive sera by chemiluminescence was higher in the group >100 GPL (19/20, 95%), as compared to that between 50 and 100 GPL (20/23, 86.9%) and that between 15 and 50 GPL (17/21, 80.9%) (Table 2). When APS sera under test were grouped according to the clinical features (Table 3) we observed that the occurrence of IgG aCL by chemiluminescence was virtually the same between patients with PAPS and patients with SAPS (p > 0.05), whereas the number of positive sera for IgM by chemiluminescence was higher in the group of PAPS as compared to SAPS patients (p b 0.001). No correlation between aCL and APS activity has been demonstrated. As expected, in the group of patients without clinical manifestations of APS, but positive for aCL (ELISA) and persistently negative for anti-β2-GPI (ELISA) and LA, the highest positivity of aCL by chemiluminescence was detected among patients
Table 2 Results of anti-CL in chemiluminescence in APS patients grouped according to the GPL values. Patients selected by ELISA
APS patients 15–50 GPL (N = 21)
Results in chemiluminescence
IgG 17/21§ 80.95%
APS patients > 100 GPL (N = 20)
APS patients 50–100 GPL (N = 23) IgM 7/21# 33.33%
IgG 20/23* 86.95%
anti-CL, anticardiolipin antibodies; APS patients, patients with antiphospholipid antibody syndrome; * p > 0.05. ^ p > 0.05. § p > 0.05. # p > 0.05.
IgM 6/23^ 26.08%
IgG 18/19*§ 94.73%
IgM 4/19^# 21.05%
A. Capozzi et al. / Journal of Immunological Methods 379 (2012) 48–52 Table 3 Results of anti-CL in chemiluminescence in APS patients. APS patients (N = 63)
PAPS (N = 15)
SAPS (N = 48)
Results in chemiluminescence
IgG 13/15* 86.66%
IgG 43/48* 89.58%
IgM 9/15^ 60%
IgM 8/48^ 16.66%
anti-CL, anticardiolipin antibodies; PAPS, patients with primary antiphospholipid antibody syndrome; SAPS, patients with secondary antiphospholipid antibody syndrome; * p > 0.05. ^ p b 0.0001.
with rheumatic disorders (50%), followed by patients with neurological disorders (33.3%) or infectious diseases (25%). All these single groups revealed a significantly lower occurrence of aCL by chemiluminescence as compared to APS patients (p b 0.0001) (Table 4). 4. Discussion In this study we tested aCL by a new automated system, using the chemiluminescence principle. Several methods have been employed for detection of aPl. However, “classical” aCL antibodies are usually present not only in APS patients, but also in patients with different autoimmune or infectious diseases (Sorice et al., 2000). Moreover, aCL have limitations according to robustness, reproducibility, standardization and clinical relevance (Devreese and Hoylaerts, 2009). On the other hand, anti-β2-GPI, as well as LA, are highly specific for APS patients, but not very sensitive. Thus, in this study, we decided to test for β2-GPI-dependent aCL selected sera obtained from two main groups of patients, by a new automated system using the chemiluminescence principle. Our results revealed a good agreement between automated chemiluminescence and ELISA in the APS patient population, according to previous data by Persijn et al. (2011). In fact, almost all the sera from APS patients, positive for IgG aCL and anti-β2-GPI, by ELISA, were also positive for IgG aCl by automated chemiluminescence. On the contrary, only 30.13% of patients without clinical manifestations of APS, but positive for aCL and negative for anti-β2-GPI (by ELISA) and LA, confirmed the positive test by chemiluminescence. Thus, automated chemiluminescence, although less sensitive than ELISA, appears to be more useful for identifying APS patients. This difference was not very surprising,
Table 4 Results of anti-CL in chemiluminescence in patients, positive for anti-CL and negative for anti-β2-GPI grouped according to the clinical features. Patients
No
No of aCL positive in chemiluminescence
%
Infectious diseases Arthritis Thrombocitopenia Pregnancy disorders Neurological disorders Gastrointestinal disorders Ocular disorders Other APS unrelated disorders
16 30 3 1 3 5 2 13
4 15 0 0 1 0 0 2
25% 50% 0% 0% 33.33% 0% 0% 15.38%
51
since this new automated system does differ from ELISA not only for the detection system, but also for antigen coating. Indeed, in our automated system a CL/β2-GPI complex was used to coat magnetic particles. Since it is well known that the antigen coating is a key element for aCL detection (Capuano et al., 2007), it is likely that the discrepancies between automated chemiluminescence and ELISA in detecting antibodies against CL may be due to the different antigenic presentation of phospholipid/β2-GPI complex on magnetic particles compared to the surface of microtitre wells. Using this method, the occurrence of aCL was significantly higher in APS patients, as compared to patients without clinical manifestations of APS, but positive for aCL by ELISA. This finding suggests that this test is able to specifically detect aPl in those patients who fulfilled the clinical criteria of APS. Interestingly, this test also prompted to identify 20% of patients positive for LA, but persistently negative for both aCL and anti-β2-GPI IgG (ELISA). This finding supports the view that aCL detection by chemiluminescence may be useful to reveal antibodies with a pathogenic role in APS. In the last few years it was demonstrated that antibodies responsible for the reaction in the aCL test also bind other negatively charged phospholipids, as well as β2-glycoprotein I (Galli et al., 1993; De Laat et al., 2004), prothrombin (Arvieux et al., 1995), protein S (Sorice et al., 1994a, 1996), protein C (Oosting et al., 1993), annexin V (Kaburaki et al., 1997), annexin II (Salle et al., 2008) and vimentin (Ortona et al., 2010). However, it is generally accepted that the combination of LAC and aCL have good correlation with the diagnosis of APS (Shoenfeld et al., 2008) and that at present routine testing of other aPL is expensive and does not significantly improve diagnosis. Taken together, our results suggest that the new technology of automated chemiluminescence assay for measuring aPL may represent a useful tool to identify “true” APS patients. References Arvieux, J., Darnige, L., Caron, C., Reber, G., Bensa, J.C., Colomb, M.G., 1995. Development of an ELISA for autoantibodies to prothrombin showing their prevalence in patients with lupus anticoagulants. Thromb. Haemost. 74, 1120. Bertolaccini, M.L., Gomez, S., Pareja, J.F., Theodoridou, A., Sanna, G., Hughes, G.R.V., Khamashta, M.A., 2005. Antiphospholipid antibody tests: spreading the net. Ann. Rheum. Dis. 64, 1639. Capuano, F., Grasso, V., Belforte, L., Pallavicini, L., Tincani, A., Andreoli, L., Bonelli, F., 2007. Development of automated assays for anticardiolipin antibodies determination: addressing antigen and standardization issues. Ann. N. Y. Acad. Sci. 1109, 493. De Laat, H., Derksen, R., De Groot, P.G., 2004. β2-glycoprotein I, the playmaker of the antiphospholipid syndrome. Clin. Immunol. 112, 161. Devreese, K., Hoylaerts, M.F., 2009. Laboratory diagnosis of the antiphospholipid syndrome: a plethora of obstacles to overcome. Eur. J. Haematol. 83, 1. Galli, M., Barbui, T., Zwaal, R.F.A., Confurius, P., Brevers, E.M., 1993. Antiphospholipid antibodies: involvement of protein cofactors. Haematology 78, 1. Gibril, F., Jensen, R.T., 1997. Comparative analysis of diagnostic techniques for localization of gastrointestinal neuroendocrine tumors. Yale J. Biol. Med. 70, 509. Hughes, G.R.V., 1985. The anticardiolipin syndrome. Clin. Exp. Rheumatol. 3, 285. Hughes, G.R.V., Harris, E.N., Gharavi, A.E., 1986. The anticardiolipin syndrome. J. Rheumatol. 13, 486. Ichikawa, K., Tsutsumi, A., Atsumi, T., Matsuura, E., Kobayashi, S., Hughes, G.R.V., Khamashta, M.A., Koike, T., 1999. A chimeric antibody with the human gamma1 constant region as a putative standard for assays to detect IgG beta2-glycoprotein I-dependent anticardiolipin and anti-beta2glycoprotein I antibodies. Arthritis Rheum. 42, 2461.
52
A. Capozzi et al. / Journal of Immunological Methods 379 (2012) 48–52
Kaburaki, J., Kuwana, M., Yamamoto, M., Kawai, S., Ikeda, Y., 1997. Clinical significance of anti-annexin V antibodies in patients with systemic lupus erythematosus. Am. J. Hematol. 54, 209. McNeil, H.P., Simpson, R.J., Chesterman, C.N., Krilis, S.A., 1990. Anti-phospholipid antibodies are directed against a complex antigen that includes a lipidbinding inhibitor of coagulation: beta 2-glycoprotein I (apolipoprotein H). Proc. Natl. Acad. Sci. U. S. A. 87, 4120. Miyakis, S., Lockshin, M.D., Atsumi, T., Branch, D.W., Brey, R.L., Cervera, R., Derksen, R.H., De Groot, P.G., Koike, T., Meroni, P.L., Reber, G., Shoenfeld, Y., Tincani, A., Vlachoyiannopoulos, P.G., Krilis, S.A., 2006. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J. Thromb. Haemost. 4, 295. Oosting, J.D., Derksen, R.H., Bobbink, I.W., Hackeng, T.M., Bouma, B.N., De Groot, P.G., 1993. Antiphospholipid antibodies directed against a combination of phospholipids with prothrombin, protein C, or protein S: an explanation for their pathogenic mechanism? Blood 81, 2618. Ortona, E., Capozzi, A., Colasanti, T., Conti, F., Alessandri, C., Longo, A., Garofalo, T., Margutti, P., Misasi, R., Khamashta, M.A., Hughes, G.R.V., Valesini, G., Sorice, M., 2010. Vimentin/cardiolipin complex as a new antigenic target of the Antiphospholipid Syndrome. Blood 116, 2960. Persijn, L., Decavele, A.S., Schouwers, S., Devreese, K., 2011. Evaluation of a new set of automated chemiluminescense assays for anticardiolipin and anti-beta2-glycoprotein I antibodies in the laboratory diagnosis of the antiphospholipid syndrome. Thromb. Res. 128, 565. Reber, G., Tincani, A., Sanmarco, M., De Moerloose, P., Boffa, M.C., 2004. Proposals for the measurement of anti-β2-glycoprotein I antibodies. Standardization Group of the European Forum on Antiphospholipid Antibodies. J. Thromb. Haemost. 2, 1860. Salle, V., Mazière, J.C., Smail, A., Cévallos, R., Mazière, C., Fuentes, V., Tramier, B., Makdassi, R., Choukroun, G., Vittecoq, O., Goëb, V., Ducroix, J.P., 2008. Anti-annexin II antibodies in systemic autoimmune diseases and antiphospholipid syndrome. J. Clin. Immunol. 28, 291. Shoenfeld, Y., Twig, G., Katz, U., Sherer, Y., 2008. Autoantibody explosion in antiphospholipid syndrome. J. Autoimmun. 30, 74.
Sorice, M., Griggi, T., Circella, A., Garofalo, T., D'Agostino, F., Pittoni, V., Pontieri, G.M., Lenti, L., Valesini, G., 1994a. Detection of antiphospholipid antibodies by immunostaining on thin layer chromatography plates. J. Immunol. Methods 173, 49. Sorice, M., Griggi, T., Circella, A., Lenti, L., Arcieri, P., Di Nucci, G.D., Mariani, G., 1994b. Protein S antibodies in acquired protein S deficiencies. Blood 83, 2383. Sorice, M., Arcieri, P., Griggi, T., Circella, A., Misasi, R., Lenti, L., Di Nucci, G.D., Mariani, G., 1996. Inhibition of protein S by autoantibodies in patients with acquired protein S deficiency. Thromb. Haemost. 75, 555. Sorice, M., Pittoni, V., Circella, A., Misasi, R., Conti, F., Longo, A., Pontieri, G.M., Valesini, G., 1998. Anti-prothrombin but not “pure” anti-cardiolipin antibodies are associated with the clinical features of the antiphospholipid antibody syndrome. Thromb. Haemost. 80, 713. Sorice, M., Pittoni, V., Griggi, T., Losardo, A., Leri, O., Magno, M.S., Misasi, R., Valesini, G., 2000. Specificity of antiphospholipid antibodies in infectious mononucleosis: a role for anti-cofactor protein antibodies. Clin. Exp. Immunol. 120, 301. Tincani, A., Balestrieri, G., Spatola, L., Cinquini, M., Meroni, P.L., Roubey, R.A., 1998. Anticardiolipin and anti-beta2 glycoprotein immunoassays in the diagnosis of antiphospholipid syndrome. Clin. Exp. Rheumatol. 16, 396. Tincani, A., Allegri, F., Balestrieri, G., Reber, G., Sanmarco, M., Meroni, P., Boffa, M.C., 2004. Minimal requirementsfor antiphospholipid antibodies ELISAs proposed by the European forum on the antiphospholipid antibodies. Thromb. Res. 114, 553. Tincani, A., Filippini, M., Scarsi, M., Galli, M., Meroni, P.L., 2009. European attempts for the standardisation of the antiphospholipid antibodies. Lupus 18, 913. Triplett, D.A., 1995. Protean clinical presentation of antiphospholipid– protein antibodies (APA). Thromb. Haemost. 74, 329. Wilson, W.A., Gharavi, A.E., Koike, T., Lockshin, M.D., Branch, D.W., Piette, J.C., Brey, R., Derksen, R., Harris, E.N., Hughes, G.R.V., Triplett, D.A., Khamashta, M.A., 1999. International consensus statement on preliminary classification criteria for definite antiphospholipid syndrome. Arthritis Rheum. 42, 1309.