Lupus anticoagulant identifies two distinct groups of patients with different antibody patterns.

BACKGROUND: Whether antibodies directed to ß2-Glycoprotein I (aß2GPI) are responsible for LA activity is not well defined. However, in the absence of such antibodies the molecule responsible for LA phenomenon is unknown. OBJECTIVE: The aim of this study was the biochemical identification of the target antigen epitope of aPL responsible of LA activity in the absence of aß2GPI antibodies together with the biological and clinical characteristics of these patients in comparison with classical triple positive patients. PATIENTS/METHODS: A comparison of patients with LA without (LA+/aß2GPI-) and those with (LA+/aß2GPI+) associated aß2GPI antibodies was performed. Size exclusion chromatography and analytical chromatography were used to identify the molecule with LA activity in patients LA+/aß2GPI-. RESULTS AND CONCLUSIONS: Analytical size-exclusion chromatography revealed a peak of 996Kd with LA activity perfectly overlapping that of IgM anti phosphatidylserine/prothrombin (aPS/PT) antibodies. Similarly, all the 25 LA+/aß2GPI- patients were positive for aPS/PT antibodies. LA+/aß2GPI- compared to 33 LA+/aß2GPI+ patients turned out to be significantly older, with a lower rate of previous thromboembolic events and a weaker LA activity. Search for aPS/PT and aß2GPI antibodies in patients with LA is useful to identify two subgroups of LA at different risk of thromboembolic events.

Clinical value of anti-domain I-ß2Glycoprotein 1 antibodies in antiphospholipid antibody carriers. A single centre, prospective observational follow-up study.

BACKGROUND: There seems to be a clear correlation between antibodies against domain I (anti-DI) of ß2Glycoprotein I and severe clinical profiles in antiphospholipid syndrome (APS) patients. We investigated the clinical significance of anti-DI antibodies in a cohort of aPL carriers. METHODS: One hundred and five carriers persistently positive for IgG anti-ß2Glycoprotein 1 antibodies (a-ß2GPI) and/or IgG anticardiolipin (aCL) and/or lupus anticoagulants (LAC) were tested for the presence of anti-DI antibodies using the QUANTA Flash® Beta2GPI-Domain I chemiluminescence immunoassay. RESULTS: Anti-DI antibodies were detected in 44 aPL carriers (41.9%) and they were significantly associated to triple aPL positivity (LAC plus IgG a-ß2GPI plus IgG aCL antibodies). Isolated LAC and a-ß2GPI antibodies were significantly associated to anti-DI negative aPL carriers. During a 82.2 month mean follow-up, ten aPL carriers (9.5%) developed a first thrombotic event so becoming APS patients. Anti-DI antibodies, triple aPL positivity, thromboembolic risk factors and autoimmune disorders significantly prevailed in carriers becoming APS. Logistic regression analysis showed that anti-DI positivity was an independent risk factor for thrombosis. CONCLUSIONS: Anti-DI antibody positivity can be considered a new risk factor predictive of the first thrombotic event in aPL carriers, instead, negative anti-DI may be useful to identify low-risk aPL carriers.

Thrombocytopenia in high-risk patients with antiphospholipid syndrome.

Essentials The prevalence of thrombocytopenia in patients with antiphospholipid syndrome is not well defined. We studied triple positive patients with antiphospholipid syndrome and its catastrophic variant. Prevalence of thrombocytopenia was 6% and 100% in patients who developed the catastrophic form. In triple positive patients thrombocytopenia is low and platelets drop during the catastrophic form. SUMMARY: Background Thrombocytopenia is the most common non-criteria hematological feature in patients with antiphospholipid syndrome (APS). This condition is more common in patients with catastrophic APS (CAPS). Objectives To evaluate the prevalence of thrombocytopenia in a large series of high-risk patients with APS, and to assess the behavior of the platelet count during CAPS. Methods/Patients This was a cross-sectional study in which we analyzed the platelet counts of a homogeneous group of high-risk APS patients (triple-positive). Six of these patients developed a catastrophic phase of the disease, and the platelet count was recorded before the acute phase, during the acute phase, and at recovery. Results The mean platelet count in 119 high-risk triple-positive patients was 210 × 109 L-1 . With a cut-off value for thrombocytopenia of 100 × 109 L-1 , the prevalence of thrombocytopenia was 6% (seven patients). No difference between primary APS and secondary APS was found. In patients who suffered from CAPS, a significant decrease from the basal count (212 ± 51 × 109 L-1 ) to that at the time of diagnosis (60 ± 33 × 109 L-1 ) was observed. The platelet count became normal again at the time of complete remission (220 ± 57 × 109 L-1 ). A decrease in platelet count always preceded the full clinical picture. Conclusions This study shows that, in high-risk APS patients, the prevalence of thrombocytopenia is low. A decrease in platelet count was observed in all of the patients who developed the catastrophic form of the disease. A decrease in platelet count in high-risk APS patients should be considered a warning signal for disease progression to CAPS.

Identification of discrete epitopes of Ro52p200 and association with fetal cardiac conduction system manifestations in a rodent model.

Congenital heart block (CHB) is a potentially lethal condition characterized by a third-degree atrioventricular block (AVB). Despite anti-Ro52 antibodies being detected in nearly 90% of mothers of affected children, CHB occurs in only 1-2% of anti-Ro/Sjögren's-syndrome-related antigen A (SSA) autoantibody-positive pregnancies. Maternal antibodies have been suggested to bind molecules crucial to fetal cardiac function; however, it remains unknown whether a single antibody profile associates with CHB or whether several specificities and cross-reactive targets exist. Here, we aimed to define further the reactivity profile of CHB-associated antibodies towards Ro52p200 (amino acid 200-239). We first analysed reactivity of a monoclonal anti-Ro52 antibody shown to induce AVB in rats (7.8C7) and of sera from anti-Ro52p200 antibody-positive mothers of children with CHB towards a panel of modified Ro52p200 peptides, and subsequently evaluated their potential to induce AVB in rats upon transfer during gestation. We observed that CHB maternal sera displayed a homogeneous reactivity profile targeting preferentially the C-terminal part of Ro52p200, in contrast to 7.8C7 that specifically bound the p200 N-terminal end. In particular, amino acid D233 appeared crucial to maternal antibody reactivity towards p200. Despite low to absent reactivity towards rat p200 and different binding profiles towards mutated rat peptides indicating recognition of different epitopes within Ro52p200, immunoglobulin (Ig)G purified from two mothers of children with CHB could induce AVB in rats. Our findings support the hypothesis that several fine antibody specificities and cross-targets may exist and contribute to CHB development in anti-Ro52 antibody-positive pregnancies.

APS - Diagnostics and challenges for the future.

Diagnosis of antiphospholipid syndrome (APS) is essentially based on the detection of circulating antiphospholipid (aPL) antibodies. Progress have been made on the standardization of tests exploring the presence of aPL as guidelines on coagulation and immunological tests were recently published in the literature. Clinical relevance of aPL profile has come from prospective cohort studies in populations with a homogeneous antibody profile supporting the view that triple positivity is a high risk pattern in patients and carriers. In addition to the classic ones, several other tests have been proposed for the diagnosis of APS. The detection of antibodies directed to domain 1 and 4/5 of ß2-Glycoprotein I (ß2GP1) were found to be particularly sound. Several issues remain to be addressed. We do not yet know what is the physiological function of ß2GP1 and the pathophysiology of thrombosis and pregnancy loss in these patients. Moreover, treatment is poorly defined especially in the case of feared catastrophic APS.

Laboratory testing for antiphospholipid syndrome.

This is a practical report on laboratory tests for the diagnosis of antiphospholipid syndrome (APS). After a general definition of APS, this study deals with appropriateness and timing in requesting the determination of antiphospholipid (aPL) antibodies. Lupus anticoagulant (LAC), anticardiolipin (aCL), and anti ß2-glycoprotein I (aßGPI) are the mandatory tests to be performed, while other tests are not yet validated for clinical use. Interpretation of results is an important discussed issue that implies a close liaison between clinical pathologists and clinicians. Finally, a personal definition of APS according to aPL antibody profile closes the manuscript.

Bedside diagnosis of two major clinical phenotypes of hypertensive disorders of pregnancy.

OBJECTIVE: To investigate the hypothesis that fetal abdominal circumference (AC) and uterine artery (UtA) Doppler pulsatility index (PI) could be used to select two homogeneous subgroups of women affected by hypertensive disorders of pregnancy (HDP), characterized by the coexistence of maternal hypertension with and without intrauterine growth restriction (IUGR). METHODS: This was a multicenter retrospective study of cases affected by HDP in whom fetal AC and UtA-PI had been measured at admission to fetomaternal medicine units. Maternal characteristics, pregnancy complications and outcome were recorded. These data allowed us to model the characteristics of fetal growth in cases affected by HDP, and to design composite indicators of risk factors for maternal metabolic syndrome and of severity for maternal functional organ damage. RESULTS: Measurements of fetal AC and UtA-PI allowed us to define a group of HDP cases with appropriate-for-gestational-age (AGA) fetuses (HDP-AGA), diagnosed by normal fetal AC and UtA-PI (n = 205), and a group of HDP cases with IUGR fetuses (HDP-IUGR), diagnosed by fetal AC < 5(th) centile and UtA-PI > 95(th) centile (n = 124). Curves fitted to the birth weights of these two groups were significantly different, but gestational age at admission for HDP (< 34 or ≥ 34 weeks) did not show an independent association with birth weight. When birth weight was expressed as a Z-score with respect to local reference charts, the average corresponded to the 6(th) and 48(th) centiles, respectively. The occurrence of HDP-AGA (as compared with HDP-IUGR) was significantly associated with risk factors for maternal metabolic syndrome (odds ratio, 2.79 (95% CI, 1.57-4.97)), independent of gestational age. The same risk factors yielded non-significant odds ratios for the development of late-onset (vs early-onset) HDP. Women with HDP-IUGR had worse clinical outcomes. CONCLUSIONS: This study provides new information based on simple prenatal bedside examinations that might help to differentiate HDP-IUGR from HDP-AGA fetuses. These groups are associated with different fetal growth patterns and risk factors, independent of gestational age at onset of the disease. Copyright © 2015 ISUOG. Published by John Wiley & Sons Ltd.

Antibodies to Domain 4/5 (Dm4/5) of ß2-Glycoprotein 1 (ß2GP1) in different antiphospholipid (aPL) antibody profiles.

BACKGROUND: Determination of the three recommended tests for the diagnosis of antiphospholipid syndrome [Lupus Anticoagulant (LA), anticardiolipin (aCL) and anti ß2-Glycoprotein 1 (aß2GP1) antibodies] allow physicians to allocate patients into classification (risk) categories. OBJECTIVES: To measure antibodies of IgG isotype directed towards Domain 4/5 (Dm4/5) of ß2GP1. PATIENTS/METHODS: In this cross-sectional study we measured IgG aß2GP1-Dm4/5 in a group of individuals positive for IgG aß2GP1 and classified as triple (LAC+, IgG aCL+, IgG aß2GP1+, n=32), double (LAC-, IgG aCL+, IgG aß2GP1+, n=23) or single positive (LA-, IgG aCL-, IgG aß2GP1+, n=10). RESULTS: Geometric mean and standard deviation of IgG aß2GP1 values expressed as Chemiluminescent Units (CU) in triple, double, single positive groups and in 40 healthy individuals were 1795±783, 321±181, 29±8 and 5.0±1.0, respectively (ANOVA p<0.0001). Geometric mean and standard deviation of IgG aß2GP1-Dm4/5 expressed as Optical Density (OD) in triple, double and single positive groups and in 40 healthy individuals were 0.16±0.13, 0.16±0.15 and 0.26±0.15, 0.13±0,11, respectively (ANOVA p<0.002). Individuals in the single positive group, expressed significantly higher values with respect to triple (p=0.04) and double (p=0.03) positive groups. Approximate OD cut-off value (99° percentile) calculated in 40 normal control subjects is 0.404. Positivity to IgG aß2GP1-Dm4/5 according to this cutoff was found in only 5 individuals, 3 in triple positive and 2 in single positive groups and was not associated with thromboembolism. CONCLUSION: Mean level of IgG aß2GP1-Dm4/5 is higher in single positive group. There is no association between positivity to IgG aß2GP1-Dm4/5 and thromboembolic events.

Are biological drugs safe in pregnancy?

The introduction of biological therapies has significantly improved the outcome of inflammatory rheumatic diseases. As most of these diseases affect women and men in childbearing age, some concerns have been voiced as to the safety of these drugs in relation to reproduction and pregnancy. Data from many hundreds of pregnancies in patients affected by inflammatory bowel disease and inflammatory arthritis have suggested that exposure to anti-TNF therapies at conception and/or during pregnancy is not associated with adverse pregnancy outcomes or any increase in congenital abnormalities. However, the exposure to anti-TNFα agents, particularly to monoclonal antibodies, in late pregnancy is associated with high drug levels in the newborn and their long-term effects on children remain unknown. Therefore, limiting the use of anti-TNFα to the first 30 weeks of pregnancy is recommended to reduce fetal exposure. Live-virus vaccines should be given only when levels of anti-TNFα drugs are undetectable in the serum of infants. Studies suggest that many of these drugs do enter breast milk in small amounts, but the extent to which the infant absorbs them is less clear. Limited reports have not suggested adverse pregnancy outcomes in women whose partners were exposed to anti-TNF therapies at the time of conception. Pregnancy data for rituximab, abatacept, anakinra, tocilizumab and belimumab are limited and their use in pregnancy cannot currently be recommended.

Antiphospholipid syndrome: antibodies to Domain 1 of ß2-glycoprotein 1 correctly classify patients at risk.

BACKGROUND: Determination of lupus anticoagulant (LA), anticardiolipin (aCL) and ß2-Glycoprotein 1 (aß2GP1) antibodies is mandatory to classify patients with antiphospholipid syndrome (APS) into risk categories. OBJECTIVES: To measure relevant antibodies, considered to be those of the IgG isotype directed towards ß2GP1 and particularly those directed to Domain 1 (Dm1) of the molecule. PATIENTS/METHODS: In this cross-sectional study we measured IgG aß2GP1-Dm1 by a chemiluminescent immunoassay in a group of individuals initially positive for IgG aß2GP1 and classified as triple (LAC+, IgG aCL+, IgG aß2GP1+, n = 32), double (LAC-, IgG aCL+, IgG aß2GP1+, n = 23) or single positive (LA-, IgG aCL-, IgG aß2GP1+, 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. The geometric mean obtained in 40 healthy subjects was 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 aß2GP1-Dm1 (above 14.2 CU) were found in 45 individuals while 20 individuals (20/65 = 30.8%) positive for IgG aß2GP1 were negative for IgG aß2GPI-Dm1. There was a significant association between positive IgG aß2GP1-Dm1 and thromboembolic events (P = 0.001). Positive and negative values of IgG aß2GP1-Dm1 were consistently confirmed after 12 weeks, with only three low positive values being negative after 12 weeks. In conclusion, IgG aß2GP1-Dm1 seems a robust and 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.

Hydroxychloroquine restores trophoblast fusion affected by antiphospholipid antibodies.

BACKGROUND: Obstetric antiphospholipid syndrome (APS) is defined by pregnancy complications associated with antiphospholipid antibodies (aPL). The mechanisms of the pathogenic effects of aPL in pregnancy are poorly understood. Toll-like receptors (TLR) have been implicated previously in APS. OBJECTIVES: The aims of our study were (1) to determine aPL effects on trophoblastic cell fusion and differentiation, (2) to identify which TLR is involved in this process, and (3) to evaluate the efficacy of hydroxychloroquine (HCQ) to counteract the effects of aPL. METHODS: BeWo cells are a model for trophoblast fusion and differentiation. Fusion index was assessed by immunocytochemical examination, and biochemical differentiation by using ELISA-measured ß-human choronic gonadotropin hormone (ß-hCG) secretion. We used three types of aPL to study their effect on cell fusion and differentiation: aPL derived from obstetric APS patients and affinity purified and polyclonal rabbit anti-ß2-glycoprotein-1 (anti-ß2GP1) antibodies. Experiments on fusion were confirmed using primary cytotrophoblastic cells. RESULTS: All of the types of aPL used decreased the fusion index in BeWo and primary trophoblastic cells (64%, 52%, and 41% for BeWo cells and 67% and 62% for primary cells, respectively), and anti-ß2GP1 antibodies decreased hCG secretion in BeWo cells (41%). To block TLR4 antibodies or to abolish TLR4 cell surface expression restored fusion index in both cell types and ß-human choronic gonadotropin hormone excretion in BeWo cells. HCQ treatment induced the same effect and decreased TLR4 mRNA (40% and 35%, respectively) and protein expressions (62% and 42%, respectively) in BeWo cells. CONCLUSION: Anti-ß2GP1 antibodies decrease trophoblastic differentiation via TLR4. This effect is restored by HCQ, suggesting its therapeutic interest in APS pregnancies.

Ferritin in the antiphospholipid syndrome and its catastrophic variant (cAPS).

BACKGROUND: Ferritin is an iron storage protein considered also as an acute phase reactant with high levels in various inflammatory conditions. Recently, a plausible role for ferritin in the pathogenesis of immune-mediated and especially autoimmune diseases has been suggested. However, the link between ferritin and the antiphospholipid syndrome (APS) has been rarely explored. Therefore, in the current study we evaluated ferritin levels and their correlation to clinical and serological manifestations in patients with APS. We further analyzed ferritin levels among patients with the catastrophic variant of APS (cAPS). METHODS: Ferritin levels were determined in serum samples of 176 APS patients and 98 matched healthy controls according to age and sex (LIAISON, DiaSorin, Italy). APS samples were further analyzed for antiphospholipid (anti-cardiolipin, anti- beta-2-glycoprotein, lupus anticoagulant) and anti-infectious antibodies (CMV, EBV, rubella, toxoplasma, HBV) (LIAISON, DiaSorin, Italy). Clinical, serological and demographic manifestations were recorded. An additional analysis of ferritin levels among 14 patients with cAPS was performed. RESULTS: Hyperferritinemia was present in 9% vs. 0% of APS patients and controls, respectively (p < 0.001). Among patients with APS, ferritin levels correlated with venous thrombosis, cardiac, neurological, and hematological manifestations and the presence of anti-CMV-IgM antibodies. Hyperferritinemia was present in 71% of cAPS patients, and ferritin levels among this subgroup were significantly higher compared with APS-non-cAPS patients (816 ± 847 ng/ml vs. 120 ± 230 ng/ml, p < 0.001). CONCLUSIONS: Herein, we found that hyperferritinemia correlates with the presence of APS, its clinical manifestations and specifically with the catastrophic variant of this disease. Hyperferritinemia was also linked with anti-CMV antibodies among patients with APS. These associations allude to a pathogenic role of ferritin in the pathogenesis of APS, and the plausible role of ferritin as a marker of ensuing cAPS, although further studies are needed to elucidate these associations.

Confirmation of initial antiphospholipid antibody positivity depends on the antiphospholipid antibody profile.

BACKGROUND: The revised classification criteria for the antiphospholipid syndrome state that antiphospholipid (aPL) antibodies (lupus anticoagulant [LAC] and/or anticardiolipin [aCL] and/or anti-ß2 -glycoprotein I [aß2 GPI] antibodies) should be detected on two or more occasions at least 12 weeks apart. Consequently, classification of patient risk and adequacy of treatment may be deferred by 3 months. OBJECTIVES: In order to early classify patient risk, we evaluated whether aPL positivity confirmation is related to aPL antibody profiles. PATIENTS AND METHODS: Consecutive patients referred to our center who were initially positive in one or more tests exploring the presence of aPL were tested after 3 months. During a 4-year period, 225 patients were initially positive in one or more tests, and 161 were available for confirmation after 3 months. Patients were classified as triple-positive (n = 54: LAC(+) , aCL(+) , aß2 GPI(+) , same isotype), double-positive (n = 50: LAC(-) , aCL(+) , aß2 GPI(+) , same isotype) and single-positive (n = 53: LAC or aCL or aß2 GPI antibodies as the sole positive test). RESULTS: Among subjects with triple positivity at initial testing, 98% (53 of 54) had their aPL profile confirmed after 12 weeks. The double-positive and single-positive groups had data confirmed in 42 of 50 (84%) and 23 of 57 (40%) subjects, respectively. CONCLUSIONS: Our results show that high-risk subjects with triple-positive aPL profiles are identified early, at the time of the initial screening tests.

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-ß2 glycoprotein I (anti-ß2GPI) 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-ß2GPI 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-ß2GPI antibodies co-incubated with platelets enhanced TRAP-6- induced platelet P-sel expression. Notably, anti-ß2GPI 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.

Correct laboratory approach to APS diagnosis and monitoring.

Triple positivity (positive Lupus Anticoagulant, anticardiolipin and anti ß2-glycoptrotein I antibodies) identifies the pathogenic autoantibody (anti Domain I of ß2-glycoptroteinI) that is present in patients with definite Antiphospholipid Syndrome (APS). This is supported by the fact that aß2GPI antibodies obtained by affinity purification in these patients possess LA activity. Moreover, patients and carriers of this profile carry a much higher risk of thrombosis and pregnancy loss than APS patients with positivity for only one of the tests. Thus, very different risk categories exist among patients with APS as well as among carriers of aPL. Clinical studies and interventional trials should first take these high risk subjects into consideration.

Interpretation of laboratory data and need for reference laboratories.

A single positive laboratory test among those exploring the presence of antiphospholipid antibodies is not associated with thromboembolic events and does not identify patients with antiphospholipid syndrome. On the other hand, more than one laboratory test positive, and in particular all three tests positive, is strongly associated to thromboembolic events and identifies high risk patients. Triple positivity is in fact related to the presence of a specific anti-ß2-glycoprotein I (anti-Domain I) antibody, also able to prolong coagulation tests. Monoclonal antibodies against Domain I with Lupus Anticoagulant activity might be candidate material for standardization of antiphospholipid assays. Much work remains to be done in this field.

Secondary prevention in thrombotic antiphospholipid syndrome.

Secondary prevention of venous thromboembolism in antiphospholipid syndrome (APS) is usually made using vitamin K antagonists (VKAs) to maintain an international normalized ratio (INR) between 2.0 and 3.0. The optimal intensity of anticoagulation was determined in two prospective randomized controlled trials, both excluding the benefit of more intense anticoagulation. The same regimen is also recommended in patients with APS and arterial thromboembolism as aspirin does not appear to protect against recurrences. The duration of treatment is usually indefinite because of a substantial risk of recurrence.