Marta Tonello

Incidence of a first thromboembolic event in asymptomatic carriers of high-risk antiphospholipid antibody profile: a multicenter prospective study

Persistent antiphospholipid (aPL) antibodies are occasionally found in subjects without prior history of thromboembolic events (TEs), raising the dilemma of whether to initiate or not a primary thromboprophylaxis. A first TE is considered rare in aPL carriers, but previous studies did not consider the aPL profile nor was the test positivity confirmed in a reference laboratory. In this study, 104 subjects with high-risk aPL profile (positive lupus anticoagulant, anticardiolipin, and anti-β(2)-glycoprotein I antibodies, triple positivity) confirmed in a reference laboratory, were followed up for a mean of 4.5 years. There were 25 first TEs (5.3% per year): the cumulative incidence after 10 years was 37.1% (95% confidence interval [CI], 19.9%-54.3%). On multivariate analysis, male sex (hazard ratio = 4.4; 95% CI, 1.5-13.1, P = .007) and risk factors for venous thromboembolism (hazard ratio = 3.3; 95% CI, 1.3-8.5, P = .01) were independent predictors for TEs. Aspirin did not significantly affect the incidence of TE. In conclusion, the occurrence of a first TE in carriers of high-risk aPL profile is considerable; it is more frequent among male subjects and in the presence of additional risk factors for venous TE. These data can help in the decision to initiate primary thromboprophylaxis in these subjects.

Risk factors for pregnancy failure in patients with anti-phospholipid syndrome treated with conventional therapies: a multicentre, case–control study

OBJECTIVEnTo identify the risk factors associated with pregnancy failure in patients with APS treated with conventional therapy.nnnMETHODSnA multicentre, case-control study was conducted to compare APS patients with successful and unsuccessful pregnancy outcomes. We retrospectively considered 410 pregnancies of women diagnosed with primary APS. The study focused on 57 unsuccessful pregnancies (considered the study population) and 57 successful pregnancies (considered the control population) matched for age and therapy. All the patients had been treated with conventional protocol treatments including low-dose aspirin and/or heparin. The clinical and laboratory features of the two groups of women diagnosed with APS were compared.nnnRESULTSnThe independent risk factors for pregnancy failure were: (i) the presence of SLE or other autoimmune diseases [odds ratio (OR) 6.0; 95% CI 1.7, 20.8; P = 0.01]; (ii) history of both thrombosis and pregnancy morbidity (OR 12.1; 95% CI 1.3, 115.3; P = 0.03); and (iii) triple [Immunoglobulin (Ig) G/IgM aCLs plus IgG/IgM anti-β(2) glycoprotein I antibodies plus LA] aPL positivity (OR 4.1; 95% CI 1.0, 16.7; P = 0.05). APS patients diagnosed on the basis of a single positive test and/or history of pregnancy morbidity alone were generally found to have successful pregnancies.nnnCONCLUSIONnIt would seem from these findings that the risk of pregnancy failure in APS women planning to conceive can be stratified on the basis of some specific clinical and laboratory features.

Antibody profile and clinical course in primary antiphospholipid syndrome with pregnancy morbidity

In women diagnosed as having category I primary obstetric antiphospholipid syndrome, clinical characteristics and the risk of subsequent thromboembolic events and further unsuccessful pregnancy has not been clearly documented. Women with unexplained obstetric complications and no definite autoimmune systemic diseases were tested for lupus anticoagulant (LA), IgG/IgM anticardiolipin (aCL) and IgG/IgM anti-human beta2-Glycoprotein I (abeta2GPI) antibodies and diagnosed as having primary antiphospholipid syndrome (APS) in classification category I on the basis of more than one laboratory criteria present in any combination. Characteristics at the time of diagnosis and risk factors for subsequent clinical events during a mean follow-up of 6.3 years were evaluated. Fifty-three of 600 women studied were found to fulfil obstetric criteria and had more than one positive laboratory test at the time of diagnosis. All the women were aCL and abeta2GPI positive, and 16 were also LA positive. This latter group (triple positivity) had distinct features and had more frequently experienced previous thromboembolism (OR = 122.5, 95% CI 16-957, p < 0.001). They also had an increased rate of late pregnancy loss (OR = 16.2, 95%CI 0.9-292, p = 0.01), and a higher IgG abeta2GPI titer at diagnosis (median, 25(th) and 75(th) percentile were 118, 37-962, vs. 23, 18-32, respectively, p < 0.0001). During follow-up, the rate of thromboembolic events was significantly higher in the group of women with triple positivity and/ or previous thromboembolism (OR = 57.5, 95% CI 2.7-1160, p = 0.0004) which were the only independent predictors of TE in the multivariate model. Recurrent pregnancy loss took place in seven out of 47 women who had a new pregnancy. Triple positivity and/or previous thromboembolism were again the only independent markers (OR = 34.4, 95% CI 3.5-335.1, p = 0.003) of an unsuccessful new pregnancy. In conclusion, in primary APS with pregnancy morbidity in classification category I, quite different groups of patients may be identified on the basis of laboratory tests. Triple positivity and/or a history of thromboembolism predict new TE events and new unsuccessful pregnancies.

Treatment of 139 Pregnancies in Antiphospholipid-positive Women Not Fulfilling Criteria for Antiphospholipid Syndrome: A Retrospective Study

Objective. The effect of low-dose aspirin (LDA) on pregnancy outcome in antiphospholipid (aPL)-positive women not fulfilling the criteria for antiphospholipid antibody syndrome (APS) was evaluated retrospectively. Methods. We evaluated 139 pregnancies of 114 aPL-positive women not fulfilling the Sydney classification criteria for definite APS (104 treated with LDA, 35 untreated). Inclusion criteria consisted of (1) any titer of aPL and no previous pregnancy or no pregnancy losses (defined as aPL carriers); (2) any titer of aPL and 1 or 2 pregnancy losses before the 10th gestational week. No women had previous thrombosis. The rate of pregnancy loss, gestational age at delivery, and birth weight percentile were compared in the treated and untreated patients. Associations between clinical and laboratory characteristics and pregnancy outcomes were investigated. Results. The rate of pregnancy loss was low in both treated and untreated groups (7.7% vs 2.9%, respectively). There were no statistically significant differences in the rate of pregnancy loss, gestational age at birth, or birth weight percentile in the treated and untreated groups. There were significant associations between gestational age at birth ≤ 34th week and positivity for lupus anticoagulant (p = 0.025) and anti-ß2-glycoprotein I IgG antibodies at titers > 99th (p = 0.016). Conclusion. LDA treatment does not appear to improve pregnancy outcome in low-risk women not fulfilling the criteria for APS. Because antibody profile seems to influence pregnancy outcome, further studies of patients stratified according to their antibody profile are warranted.

The clinical performance of a chemiluminescent immunoassay in detecting anti-cardiolipin and anti-β2 glycoprotein I antibodies. A comparison with a homemade ELISA method

Abstract Background: Fully automated chemiluminescence immunoassays (CLIAs) are emerging technologies for the detection of anti-cardiolipin (aCL) and anti-β2 glycoprotein I (anti-β2GPI) antibodies for anti-phospholipid syndrome (APS) classification, which is commonly based on an enzyme-linked immunosorbent assay (ELISA) test result. CLIA and a homemade ELISA were used in this study to detect these antibodies, and their performances were compared. Methods: Sera were collected from 104 patients with primary APS, 88 seronegative subjects who met the clinical but not the laboratory criteria for APS, and 150 control subjects. IgG/IgM aCL and IgG/IgM anti-β2GPI antibodies were determined in the sera using a CLIA (HemosIL AcuStar®) and a homemade ELISA. Results: CLIA had a significantly lower comparative sensitivity for IgM aCL and IgG/IgM IgG anti-β2GPI antibodies; its comparative specificity was higher with respect to ELISA for IgM aCL and IgM anti-β2GPI antibodies. The two techniques showed a high, significant agreement (p<0.001) and a significant titer correlation (p<0.001). CLIA also detected IgG/IgM aCL and IgG anti-β2GPI antibodies in the seronegative patients. There was a significantly higher prevalence of IgG aCL and IgG anti-β2GPI antibodies (p<0.001 and p=0.01, respectively) in those patients with respect to that in the control population. Conclusions: Despite a lower comparative sensitivity, CLIA showed a higher comparative specificity for some aPL and a good level of agreement and correlation with a homemade ELISA. CLIA also detected some aCL and anti-β2GPI antibodies in the seronegative patients not usually identified by homemade ELISA.

Platelet and endothelial activation in catastrophic and quiescent antiphospholipid syndrome.

Antiphospholipid antibodies (aPL) seem to induce a prothrombotic state by activating endothelium and platelets, but no studies have evaluated systematically the effects of aPL from patients with the antiphospholipid syndrome (APS) in quiescent versus catastrophic phase. Our aims were to evaluate the in vitro effects on platelet activation of anti-β2 glycoprotein I (anti-β2GPI) antibodiesisolated from APS patientin either quiescent or catastrophic phase and to investigate ex vivo platelet and endothelial activation in patients with quiescent or catastrophic APS. Anti-β2GPI antibodies were isolated from plasma of a pregnant woman in two different stages of APS (quiescent and catastrophic, respectively). They were co-incubated with washed platelets from healthy controls that were then challenged with TRAP-6 (thrombin receptor activating peptide 6) and the expression of P- selectin (P-sel) on platelets was assessed by flow cytometry. Moreover, plasma samples from six patients with quiescent, four with catastrophic APS and 10 controls were assessed for several markers of platelet and endothelial activation. The results showed that purified anti-β2GPI antibodies co-incubated with platelets enhanced TRAP-6- induced platelet P-sel expression. Notably, anti-β2GPI antibodies isolated during the catastrophic phase enhanced platelet P-sel expression more than antibodies isolated from the same patient in the quiescent stage of disease. Moreover, APS patients had significantly higher plasma levels of soluble (s) Psel, sCD40 ligand, soluble vascular cell adhesion molecule 1 and monocyte chemoattractant protein 1 than control subjects. In addition, sP-sel and von Willebrand factor activity were significantly higher during catastrophic than in quiescent phase.

Relationship between antiphosphatidylserine/prothrombin and conventional antiphospholipid antibodies in primary antiphospholipid syndrome.

Abstract Background: Antiphosphatidylserine/prothrombin complex (aPS/PT) antibodies are emerging as an important marker for antiphospholipid syndrome (APS). We aimed to compare their performance with that of conventional antiphospholipid antibodies (aPL) such as lupus anticoagulant (LA), anticardiolipin (aCL), and anti-β2-glycoprotein I (anti-β2GPI) in APS and to assess their frequency in APS-negative (APS-ne) patients. Methods: We considered 160 APS patients and 128 APS-ne patients with clinical criteria for APS but tested negative for conventional aPL. Immunoglobulin (Ig)G/IgM aPS/PT, IgG/IgM aCL, and IgG/IgM anti-β2GPI were detected using ELISA assay and LA with a series of coagulation tests. Results: IgG aPS/PT were significantly associated with IgG aCL, IgG anti-β2GPI, and LA (p<0.0001 for all). IgM aPS/PT were significantly associated only with LA (p<0.0001) instead. There was a significant correlation between IgG aPS/PT and both IgG aCL and IgG anti-β2GPI levels (ρ=0.42 and ρ=0.40, respectively). Both IgG aPS/PT and IgM aPS/PT positivity significantly correlated with LA (ρ=0.44 and ρ=0.5, respectively). IgG and IgM aPS/PT were significantly more frequent in triple than in double and in single positivity (p<0.0001). According to multivariate analysis, IgG and/or IgM aPS/PT were independent risk factors for LA. APS/PT antibodies were found in 9.4% of the APS-ne patients vs. 2% of healthy control (p=0.043); those antibodies were significantly more frequent in the thrombosis with respect to the pregnancy morbidity subset (p=0.01). Conclusions: Our data attribute a clinical relevance to both IgG and IgM aPS/PT antibodies. In particular, the significant prevalence of aPS/PT in APS-ne patients suggests including them as additional laboratory criterion for APS.

IgA anticardiolipin and IgA anti-β2 glycoprotein I antibody positivity determined by fluorescence enzyme immunoassay in primary antiphospholipid syndrome.

Abstract Background: Primary antiphospholipid syndrome (PAPS) is an autoimmune disease characterized by thrombosis and/or pregnancy morbidity as well as blood antiphospholipid (aPL) antibodies such as anticardiolipin (aCL), anti-β2 glycoprotein I (anti-β2GPI) antibodies of the IgG/IgM isotype and lupus anticoagulant (LA). The clinical significance of aCL and anti-β2GPI antibodies of the IgA isotype in PAPS is still a controversial issue. Methods: Sera and plasma were collected from 84 PAPS patients (54 with thrombosis and/or pregnancy morbidity and 30 with pregnancy morbidity alone), 66 seronegative patients (subjects with clinical manifestations of PAPS although with negative results on conventional antiphospholipid antibody testing), and 78 healthy blood donors. IgA aCL and IgA anti-β2GPI were determined using fluorescence enzyme immunoassay (FEIA), (EliATM, Phadia AB, Uppsala, Sweden). For comparison purposes, the sera were also tested for IgG/IgM aCL/anti-β2GPI antibodies using the same immunoassay method. LA was assayed following internationally accepted guidelines. Results: Present respectively in 19% and 50% of the PAPS patients studied, IgA aCL and IgA anti-β2GPI antibody frequencies were both statistically significant (p=0.001 and p<0.001, respectively). The mean titers of both IgA aCL and IgA anti-β2GPI antibodies were higher in the thrombotic patients, but only the latter were significantly associated with thrombosis. Isolated IgA anti-β2GPI antibody positivity was significantly prevalent (p=0.04) in seven (10.6%) of the seronegative patients. Conclusions: Positivity to IgA anti-β2GPI antibody detected using FEIA was found to be clinically relevant in PAPS patients. Moreover the prevalence of isolated IgA anti-β2GPI antibody positivity was significant in the seronegative patients.

Longterm Outcome of Patients with Primary Antiphospholipid Syndrome: A Retrospective Multicenter Study

Objective. To assess the longterm frequency of thrombotic recurrences, obstetrical complications, organ damage, severe comorbidities, and evolution toward connective tissue disease (CTD) in primary antiphospholipid syndrome (PAPS). Methods. Medical records of patients with PAPS followed in 6 centers for ≥ 15 years were retrospectively reviewed. Results. One hundred fifteen patients were studied: 88% women, followed between 1983 and 2014 with a mean (± SD) age at diagnosis of 33 (± 10) years. During a median followup of 18 years (range 15–30), 50 patients (44%) had at least a thrombotic event for a total of 75 events and an annual incidence of 3.5%. Thromboses were more frequent in patients with previous thrombotic history (p = 0.002). A catastrophic antiphospholipid syndrome occurred in 6 patients (5%). The use of oral anticoagulants in patients with thrombotic onset did not appear to be protective against recurrences (p = 0.26). Fifty-two women had 87 pregnancies, successful in 78%. Twenty-nine percent of patients accrued functional damage. Damage was significantly associated with a thrombotic history (p = 0.004) and with arterial events (p < 0.001), especially stroke, but not with demographics, serology, or treatment. Twenty-four major bleeding episodes were recorded in 18 patients, all receiving anticoagulants. Severe infections affected 6 patients (5%), with 1 fatality. A solid cancer was diagnosed in 8 patients (7%). Altogether, 16 patients (14%) developed an autoimmune disease and 13 (11%) a full-blown picture of CTD. Conclusion. Despite therapy, a high proportion of patients experienced new thrombotic events and organ damage, while evolution toward CTD was infrequent.

Antiphosphatidylserine/prothrombin antibodies as biomarkers to identify severe primary antiphospholipid syndrome.

Abstract Background: Anti-phosphatidylserine/prothrombin (aPS/PT) antibodies have begun to be considered potentional biomarkers for antiphospholipid syndrome (APS). This cohort study investigate the role of aPS/PT antibodies as a risk factor for severe APS by evaluating the association between those antibodies and clinical/laboratory profiles of APS. Methods: Plasma/serum samples from 197 APS patients, 100 healthy subjects and 106 patients with autoimmune diseases were collected. IgG/IgM aPS/PT antibodies were assayed using commercial ELISA kit. Results: Prevalences of IgG and IgM aPS/PT (p<0.0001 and p=0.0009, respectively) and their titres (p<0.0001 and p=0.0002, respectively) were significantly higher in thrombosis/pregnancy group with respect to pregnancy morbidity alone. Prevalences of IgG and IgM aPS/PT (p<0.0001 and p=0.0004, respectively) and their mean levels (p=0.0001 for both) were significantly higher in the prematurity linked to life-threatening obstetric complications group with respect to miscarriage group. There was a significant relationship between IgG and IgM aPS/PT (p=0.001 and p=0.0002) and their mean levels were higher (p=0.0004 and p=0.0002, respectively) in the thrombotic microangiopathy group, considered a milestone manifestation of catastrophic APS. The relationship between IgG and IgM aPS/PT was significant and mean levels were higher in triple positive antiphospholipid antibody patients than in double and single positivity ones (p<0.0001 for all). Conclusions: APS/PT antibodies were associated to severe thrombosis, severe pregnancy complications inducing prematurity, and vascular microangiopathy, all generally associated to high risk APS forms requiring strong therapy.