Antibodies to Domain 1 ( Dm 1 ) of β 2-Glycoprotein 1 ( β 2 GP 1 ) correctly classify patients at risk in Antiphospholipid Syndrome ( APS )

Background. Determination of Lupus Anticoagulant (LA), anticardiolipin (aCL) and β2-Glycoprotein 1 (aβ2GP1) antibodies is mandatory to classify patients with antiphospholipid syndrome (APS) into risk categories. Objectives. To measure relevant antibodies considered those of IgG isotype directed towards β2GP1 and particularly those directed to Domain 1 (Dm1) of the molecule. Patients/Methods. In this cross-sectional study we measured IgG aβ2GP1-Dm1 by a chemiluminescent immunoassay in a group of individuals initially positive for IgG aβ2GP1 and classified as triple (LAC+, IgG aCL+, IgG aβ2GP1+, n=32), double (LAC–, IgG aCL+, IgG aβ2GP1+, n=23) or single positive (LA-, IgG aCL-, IgG aβ2GP1+, n=10). Results and Conclusion. Geometric mean and standard deviation expressed as Chemiluminescent Units (CU) in triple, double and single positive groups were 273.0 ± 6.2, 18.2 ± 9.6 and 4.4 ± 2.2, respectively. Geometric mean obtained in 40 healthy subjects is 2.0 ± 2.0. Mean CU values were significantly different among groups and with respect to values found in 40 healthy subjects (p<0.0001). Positive values of IgG aβ2GP1-Dm1 (above 14.2 CU) were found in 45 individuals while 20 individuals (20/65=30.8%) positive for IgG aβ2GP1 were negative for IgG aβ2GPI-Dm1 . There was a significant association between positive IgG aβ2GP1-Dm1 and thromboembolic events (p=0.001). Positive and negative values of IgG aβ2GP1-Dm1 were consistently confirmed after 12 weeks with only 3 low positive values resulting negative after 12 weeks. In conclusion, IgG aβ2GP1-Dm1 seems a robust and A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. reproducible test that in association with the classic tests may be useful in clinical practice in identifying individuals at high risk of developing thromboembolic events. Introduction Antiphospholipid Syndrome (APS) is characterized by the presence of antiphospholipid (aPL) antibodies in patients with thrombosis or pregnancy morbidity (1). To categorize APS patients, three tests exploring the presence of aPL antibodies [Lupus Anticoagulant (LA), anticardiolipin (aCL) and anti-β2-Glycoprotein 1 (aβ2GP1) antibodies] are performed. In this way, APS patients are classified into category I when more than one test is positive, and IIa IIb and IIc according to the presence of only one positive test (1). When all three tests are positive (triple positivity) the association with thrombosis is very strong; it gets weaker in double positive (with LA negative) and single positive patients (2). Antibodies determining positivity in all the three tests are those directed against β2GP1, a plasma protein without a defined function (3, 4). Indeed, affinity purified IgG aβ2GP1 spiked into normal plasma made all three tests positive (3). Moreover, a subgroup of IgG aβ2GP1, those directed to Domain1 (Dm1) in the NH2 terminal region of the molecule, are able to interfere with the coagulation process and are associated with thrombosis (5). The aim of this cross-sectional study was to measure both basal and after 12 weeks values of IgG aβ2GP1-Dm1 in individuals initially positive for IgG aβ2GP1. Material and Methods Study population Consecutive individuals referred to Rheumatology out-patients clinic dedicated to APS patients and initially positive for aPL antibodies were evaluated. The selection criteria for individuals were A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. not based on the diagnosis of the patients, but rather on the presence of APS-related symptoms (thrombosis, pregnancy morbidity) and aPL antibody positivity. Since IgG aβ2GP1-Dm1 is a subgroup of IgG directed to β2GP1, the study focused on the group of individuals initially positive for IgG aβ2GP1. They were classified into three groups: triple positive (LA+, IgG aCL+, IgG aβ2GP1+), double positive (LA–, IgG aCL+, IgG aβ2GP1+), and single positive (LA-, IgG aCL-, IgG aβ2GPI1+). Two of us (VP and AR) consulted the available hospital records and diagnosed previous thromboembolic events only in the presence of objective criteria and if confirmed by positive compression ultrasonography or venography for deep vein thrombosis, and spiral tomography, ventilation-perfusion lung scan or pulmonary angiography for pulmonary embolism. Positive tomographic scanning, magnetic resonance imaging, or angiography confirmed the diagnosis of arterial thromboembolism. The presence of pregnancy morbidity according to Miyakis (1) was assessed by individual interview. Hospital records were also consulted for the presence of associated autoimmune diseases (SLE or other connective tissue disease). No subject was lost to follow up after 12 weeks. We restricted this study to individuals positive for IgG aβ2GP1 but we have also tested IgG aß2GP1-Dm1 in individuals negative for IgG aβ2GP1 but positive for one or more tests exploring the presence of aPL. Determination of aPL antibodies Venous blood was collected in 3.8% sodium citrate (9:1) and centrifuged twice at 2000g for 15 min at 4 °C and obtained plasma was stored at − 80 °C un l used. Patients’ plasma were screened for the presence of aPL antibodies using home-made IgG and IgM aCL ELISA as previously described (6) using the recommendations proposed by a task force during the 13 International Congress on Antiphospholipid Antibodies, Galveston, TX (7). LA was detected using dRVVT and SCT A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. (Instrumentation Laboratory, Milan, Italy), according to the ISTH guidelines (8). The presence of IgG aβ2GP1 antibodies was confirmed by a chemiluminescent immunoassay (QUANTA flash β2GPI IgG, Inova Diagnostics, San Diego, CA, USA). The cut-off value of tests exploring the presence of aPL antibodies was calculated by means of the 99 percentile of results obtained in 40 healthy subjects. Cut-off value for both dRVVT and SCT in mixing studies was 1.2; those of IgG and IgM aCL and IgG and IgM aβ2GP1 were 10 GPL and 8 MPL and 13 and 7 home units, respectively. IgG aβ2GP1 cut-off value in chemiluminescent immunoassay was 19.4 CU. Determination of IgG aß2GP1-Dm1 antibodies Autoantibodies to β2GP1-Dm1 were measured by a chemiluminescent immunoassay (QUANTA Flash Domain 1 IgG, Inova Diagnostics, San Diego, CA, USA) using the BIO-FLASH® technology (Inova Diagnostics, San Diego, CA, USA). Plasma sample was diluted 1:10 by the instrument in a disposable plastic cuvette. The technician performing the assay was blinded to patients/individuals classification categories. Small amounts of the diluted patient plasma and the assay buffer were all combined into a second cuvette containing purified recombinant β2GP1-Dm1 coated onto paramagnetic beads, mixed and incubated at 37°C. Isoluminol conjugated anti-human IgG antibody was then added to the cuvette, and incubated at 37°C. The light coming from the luminescent reaction produced by isoluminol conjugate was measured as Relative Light Units (RLU) by the BIO-FLASH® optical system. The RLU are proportional to the amount of bound isoluminol conjugate, which in turn is proportional to the amount of aβ2GP1-Dm1 antibodies bound to the β2GP1-Dm1 on the beads. Relative light units are converted to chemiluminescent units (CU) when the assay is assigned to a lot-specific master curve, and this curve is calibrated with QUANTA Flash calibrators to obtain the final CU values for results. The approximate cut-off A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. value for IgG aβ2GP1-Dm1, calculated by means of the 99 percentile of results obtained in 40 healthy subjects was 14.2 CU. Intra-assay and inter-assay co-efficient of variation are 6.3% and 2.0%, respectively.