Patients with antiphosholipid syndrome and thrombotic recurrences: A real world observation (the Piedmont cohort study).

BACKGROUND: Patients with antiphospholipid syndrome (APS) often have thrombotic recurrences, sometimes despite appropriate ongoing anticoagulant treatment. Identifying APS vascular patients at high risk for thrombotic recurrences is still an unsolved issue. OBJECTIVES: To report the real-life experience of thrombotic recurrences in APS patients included in the Piedmont observational cohort study, and evaluate clinical and laboratory risk factors for thrombotic recurrences. PATIENTS: A multi-centre observational study was performed by enrolling 177 patients with vascular APS (primary APS in 99 subjects (56%)); the median follow-up was five years (range 1-26 years). RESULTS: The observed thrombotic recurrence rate was about 7.5/100 patient years in the first five years after the first thrombotic event. While the first recurrence often occurred (45%) in patients who were not on oral anticoagulant therapy (OAT), the second recurrence mainly occurred despite ongoing OAT (80%). However, due to the real-life observational nature of this study, treatment was based on the treating physician's judgement, and no structured therapeutic protocol was applied. Moreover, compliance with OAT was not available. No differences in antiphospholipid antibodies (aPL) profile were observed between patients with or without thrombotic recurrences, but a high risk aPL profile (Miyakis type 1 and 2a) was present in 96% of our patients, 26% of whom had triple positivity. Diabetes (p < 0.01, OR 10), inherited thrombophilia (p < 0.0078, OR 4) and OAT withdrawal were independent risk factors for recurrences. CONCLUSIONS: With the limit of a real-life observational cohort study, the thrombotic recurrence rate in APS was as high as 7.5/100 patient years in the first five years after the first thrombotic event. OAT discontinuation, diabetes and inherited thrombophilia, when associated with a high-risk aPL profile, are risk factors for thrombotic recurrences.

Antiphospholipid syndrome in northwest Italy (APS Piedmont Cohort): demographic features, risk factors, clinical and laboratory profile.

We report the experience from the Antiphospholipid Antibodies (aPL) Regional Consortium in northwest Italy, meant to support clinical research and foster collaboration among health professionals regarding the diagnosis and management of antiphospholipid syndrome (APS) patients. This cohort-study (APS Piedmont Cohort) was designed to register the clinical characteristics at inception and associated immunological manifestations at diagnosis (if any) of patients who strictly fulfilled the current criteria for APS, all recruited at the Piedmont and Valle d'Aosta regions. Clinical and laboratory data from 217 APS patients (171 with vascular events, 33 with pregnancy morbidity and 13 with both), from 16 centres within the geographical area were collected. Venous thrombosis was recorded in 45.6% of patients, arterial thrombosis in 35%, small-vessel thrombosis in 1.12% and mixed arterial and venous thrombosis in the remaining 19.4% of the cases. Pregnancy morbidity included 19 patients with unexplained fetal death beyond the 10th week of pregnancy, 17 with premature birth before the 34th week and 10 with three or more unexplained spontaneous abortions before the 10th week of gestation. This consortium represents an instrument by which to audit clinical practice, to provide counselling to local centres and to sustain future basic and clinical APS research.

Anti-cardiolipin and anti-ß2-glycoprotein I antibodies: normal reference ranges in northwestern Italy.

Laboratory tests for anticardiolipin antibodies (aCL) and anti-ß2glycoprotein I antibodies (a-ß2GPI) face problems common to many autoantibody assays: the lack of a reference standard and the need for each laboratory to assess assay-specific cut-off values. The aims of the study were to evaluate the reference range upper limits (99th percentile) used for aCL and a-ß2GPI in the northwest of Italy and to investigate the analytical performances of these assays with the newly obtained reference ranges. We assayed aCL and a-ß2GPI in 104 serum samples from patients without a history of thrombosis, pregnancy morbidity, tumours, infections and/or autoimmune diseases (30 males and 74 non-pregnant females). We tested all the commercial assays available in our regions (i.e. Orgentec Diagnostika, Aesku Diagnostics and Inova Diagnostics ELISA; CliA Zenit-RA and EliA Phadia Laboratory Systems). A further 30 serum samples, including 10 from healthy subjects, 10 from antiphospholipid syndrome (APS) patients and 10 from septic patients were assessed to investigate the analytical performance of the obtained cut-off limits. Reference range upper limits obtained with the commercial kits differ among assays and from the values reported by the manufacturer. Moreover, normal reference ranges calculated for IgG and IgM aCL differed from the arbitrary selected laboratory classification values suggested in the guidelines of 40 GPL and MPL.

Survey of lupus anticoagulant diagnosis by central evaluation of positive plasma samples.

OBJECTIVE: To determine whether the diagnosis of lupus anticoagulant (LAC) in a large cohort of positive patients was confirmed at a reference laboratory. METHODS: Over a 1-year period, each participating center collected samples from LAC-positive patients. Plasma was filtered and kept deep-frozen until it was sent on dry ice to the reference laboratory by express courier. Centers returned detailed laboratory information and clinical data from each patient. The reference laboratory screened plasma samples by diluted Russell viper venom time (dRVVT) and kaolin clotting time (KCT). When these were prolonged, 1:1 mixing studies were carried out, and confirmatory tests were performed as appropriate. Positive samples were further tested by thrombin time (TT). The presence of heparin was checked by measuring antifactor Xa activity when TT was prolonged. Negative samples were tested by activated partial thromboplastin time using hexagonal phospholipids. RESULTS: Plasma samples from 302 patients from 29 anticoagulation clinics were analyzed. LAC was excluded in 71 samples (24%), because dRVVT and KCT screening test results were normal (34) or reversed to normal by mixing studies (35). The remaining two samples were considered negative because they contained heparin. LAC-negative patients showed different characteristics from those in whom diagnosis was confirmed. They were significantly older (49.7 vs. 45.0 years, P < 0.03), were more often first diagnosed (66% vs. 41%, P < 0.001), and were more frequently judged as mild in LAC potency (60% vs. 25%, P < 0.0001). Moreover, anticardiolipin and anti-beta(2)-glycoprotein I antibody values were more often normal in LAC-negative (82%) than in LAC-positive (42%) samples (P < 0.0001). LAC-positive samples identified by both dRVVT and KCT (146/231, 63%) showed a LAC potency that was significantly stronger than that in samples in which LAC diagnosis was made by a single test. CONCLUSIONS: A false-positive LAC diagnosis is not uncommon across specialized centers. Patients' characteristics and a complete antiphospholipid antibody profile may help to identify these individuals.

Analytical and clinical comparison of different immunoassay systems for the detection of antiphospholipid antibodies.

INTRODUCTION: We evaluated analytical and clinical performances of IgG and IgM anticardiolipin (aCL) antibodies and anti-ß2-glycoprotein I (a-ß2GpI) antibodies and upper limit reference ranges (99th percentiles) in comparison with manufacturer's cutoff values with different commercial methods. METHODS: We assayed aCL and a-ß2GpI in serum samples from 30 healthy individuals, 77 patients with antiphospholipid syndrome (APS) diagnosed according to the Sydney criteria, 51 patients with autoimmune diseases, eight patients with previous thrombotic events, six patients with other diseases, and 18 patients with infectious diseases, using ELISA Inova Diagnostics; EliA Phadia Laboratory Systems; CliA Zenit-RA; and CliA Bio-Flash. RESULTS: Anticardiolipin and a-ß2GpI IgG and IgM immunoassays showed good analytic performances with both 99th percentile and manufacturer's cutoff reference values. Our results showed fair to moderate agreement among assays. In-house cutoff values gave significantly better performances only for a-ß2GpI IgG with all the immunoassays analyzed with the exception of Inova CliA Bio-Flash where we obtained the same performances with in-house and manufacturer's cutoffs. CONCLUSIONS: By guidelines, all laboratories are strongly advised to validate/verify the manufacturer's cutoff values. We recommend establishing low-positive, medium-/high-positive, and high-positive CliA IgG aCL and a-ß2GpI ranges in order to help clinicians in the diagnosis and treatment of APS.

Use of modified functional assays for activated protein C resistance in patients with basally prolonged aPTT.

Inherited resistance to activated protein C (APCr) is currently recognized as the most prevalent cause underlying venous thrombophilia, with an estimated prevalence around 20% in thrombotic patients and around 1.8-7% in the general population. A correct laboratory diagnosis of APCr is therefore essential. Two different diagnostic approaches are at present at our disposal: the semi-quantitative plasma test based on the measurement of two aPTTs (in the presence and absence of activated protein C), and the detection of the factor V Arg506 Gln mutation by DNA analysis. In this study we firstly evaluated sensitivity, specificity and diagnostic efficiency of an aPTT-based plasma clotting test (Chromogenix, Sweden) versus DNA analysis; then, since the APC resistance test is invalidated by a basally prolonged aPTT (i.e. during warfarin and heparin therapy or in patients with clotting factor deficiencies or in the presence of a lupus anticoagulant), patient plasmas were conveniently diluted in factor V deficient plasma in order to correct clotting factor abnormalities. Nevertheless, patients with a LA and an aPTT ratio range 1.8-3.17 were still all misclassified. We obtained correct diagnoses in LA positive patients by preincubating plasmas with a mixture of phospholipids; therefore we decided to perform a double modified clotting test adding a mixture of platelet derived phospholipids to samples previously diluted in factor V deficient plasma. The performance characteristics of this novel method with a different aPTT reagent (Behring, Germany) were also evaluated. With this double modified test all patients were correctly classified as negative or positive for factor V mutation in agreement with DNA analysis, irrespectfully of the basal aPTT value and the aPTT reagent employed. We propose this modified version of the APCr clotting test as an easily reproducible, reliable, very sensitive and specific screening test which possibly reduces the need for DNA analysis.

Lupus anticoagulant and thrombosis: role of von Willebrand factor multimeric forms.

OBJECTIVE: Patients with lupus anticoagulant (LA) have an increased incidence of venous and arterial thrombosis whose pathogenesis is still unclear. High molecular weight von Willebrand Factor (vWF) multimers seem to play a causal role in shear stress-induced platelet aggregation and thrombus formation. We studied whether in patients with LA, alterations in the vWF multimers might coexist. METHODS: The multimeric composition of plasma vWF was analysed by SDS-electrophoresis and immunoblotting in 43 subjects positive for LA. About 2/3 of the patients had had either ischemic stroke, recurrent abortions, deep vein thrombosis (DVT) or a combination of these; the remaining subjects had never had any thrombotic events. RESULTS: An abnormal vWf multimeric pattern was found in 16 patients (37.2%); no correlation was found with the diagnosis, but the presence of abnormal vWF significantly correlated with the site of the thrombosis: indeed, it was never detected in subjects with DVT, but was found in 71.4% of patients with multiple abortions, in 50% of those with stroke and even in 25% of non-thrombotic patients. CONCLUSION: The hypothesis is put forward that abnormal VWF may represent an additional risk factor to LA for arterial thrombosis.

Thrombotic recurrences and bleeding events in APS vascular patients: a review from the literature and a comparison with the APS Piedmont Cohort.

In APS vascular patients, thrombotic recurrences are more frequent than in non-APS thrombotic patients. To better define this clinical setting, a systematic review of the literature after 1999 was performed: 8 cohort studies (including the recent APS Piedmont Cohort) and 6 intervention studies were selected and evaluated. Thrombotic recurrences, bleeding events, therapeutic strategies, antiphospholipid (aPL) profile, inherited and acquired risk factors (when present) were calculated and compared. Emerging risk factors for thrombotic recurrences include withdrawal of oral anticoagulant therapy (OAT), high intensity OAT (INR range 3-4), aPL profile (triple positivity, Miyakis types 1 and 2a profiles) and association with inherited or acquired pro-thrombotic risk factors. Moreover, there are evidences that high risk (mainly for aPL profile) APS vascular patients have a high recurrence rate in spite of correct OAT treatment. Clinical trials in this clinical setting are needed.

Prevalence and significance of anti-prothrombin (aPT) antibodies in patients with Lupus Anticoagulant (LA).

OBJECTIVE: Anti-prothrombin (aPT) antibodies have been found in Lupus Anticoagulant (LA) positive patients. Their prevalence and relative contribution to thromboembolic risk in LA-positive patients is not well defined. The aim of this study was to determine their presence and association with thromboembolic events in a large series of patients with confirmed LA. METHODS: Plasma from LA-positive patients was collected at Thrombosis Centers and sent to a reference central laboratory for confirmation. Positive plasma was tested using home-made ELISA for the presence of aPT and anti-beta(2)GPI antibodies. RESULTS: LA was confirmed in 231 patients. Sixty-one of 231 (26%, 95%CI 22-33) LA positive subjects were positive for IgG aPT and 62 (27%, 95% CI 21-33) were positive for IgM aPT antibodies. Clinical features of Antiphospholipid Syndrome (APS) were not associated with the presence of IgG aPT [43 APS in 61 (70%) positive and 109 APS in 170 (64%) negative IgG aPT subjects, p=ns] or IgM aPT. Rate of positivity of IgG and IgM a beta(2)GPI was significantly higher than that of IgG and IgM aPT. Clinical events accounting for APS occurred in 97 of 130 (75%) IgG a beta(2)GPI positive and in 55 of 101 (54%) IgG a beta(2)GPI negative patients (OR 2.4, 95% CI 1.4 to 4.3, p=0.002). No significant association with clinical events in patients positive for both IgG aPT and IgG a beta(2)GPI as compared to those positive for one or another test was found. When patients negative for both IgG aPT and IgG a beta(2)GPI (LA positive only) were compared with remaining patients, a significantly lower association with clinical events was found (OR=0.4, 95% CI: 0.2 to 0.7, p=0.004). CONCLUSIONS: As compared to IgG a beta(2)GPI, the prevalence of IgG aPT in patients with LA is significantly lower and not associated with the clinical features of APS.

Antiphospholipid antibodies and hypertension.

Hypertension is a common manifestation of antiphospholipid syndrome (APS). Antiphospholipid antibodies (aPL) have been described in patients with hypertension secondary to renal artery stenosis (RAS). Twenty-six patients with RAS and 25 patients with severe essential hypertension (diastolic blood pressure > 110 mmHg or > or = 3 hypertensive drugs) were studied and compared to 61 age- and sex-matched healthy subjects. Serum samples were tested for lupus anticoagulant (LA), anticardiolipin (aCL) IgG and IgM, antiprothrombin (aPT) IgG and IgM, anti-beta2glycoprotein 1 (abeta2GP1) IgG and IgM. aPL were negative in all patients with RAS. Two patients with essential hypertension had positive aPL (8%) (LA in one patient confirmed in a second assay and abeta2GP1-IgG in the other patient confirmed one year later together with aCL IgG positivity). Among healthy subjects, one case (1.6%) was found to be positive for LA, aCL IgM, abeta2GP1 IgM, aPT IgG, aPT IgM. In conclusion, the association between RAS and aPL seems to be casual rather than an expression of an elective thrombotic localization ofAPS. The positive finding of aPL in 8% of patients with essential hypertension, a frequency higher than that of the control population, deserves further studies in larger series to better explore the relationship between aPL and hypertension.

Factor V Arg506-->Gln mutation in patients with antiphospholipid antibodies.

Antiphospholipid antibodies (APA) have thought to be implicated in the pathogenesis of both arterial and venous thrombosis. Because of heterogeneity of APA, direct evidence of their involvement in a thrombotic event is not yet available. Development of thrombosis in the antiphospholipid antibody syndrome (APS) may occur because of the presence of additional risk factors. Here we have analysed 60 patients with APA for the presence of the Arg506-->Gln mutation in factor V. Among them 26 suffered from deep venous thrombosis, 13 from arterial thrombosis and 21 had no history of arterial or venous thrombosis. In the first group four patients were found to be heterozygous and one homozygous for the factor V Arg506-->Gln mutation. None of the patients with the factor V mutation was found in the second and third group. The incidence of factor V mutation was significantly elevated in the group of patients with venous thrombosis. These data suggest that in patients with antiphospholipid antibodies the factor V Arg506-->Gln mutation may play a major role in the occurrence of venous thrombosis.

Arterial and venous thrombosis in two Italian families with the factor V Arg506-->Gln mutation.

APC resistance, due to a point mutation in factor V at amino acid position Arg506, has been identified as a major cause of inherited thrombophilia. Here we report the presence of the factor V Arg506-->Gln mutation in 2 Italian families. In 1 family 3 subjects heterozygous and 2 subjects homozygous for the factor V Arg506-->Gln mutation were identified. The only subject who developed a thrombotic event was a 20-yr-old girl who was found to be homozygous for the factor V Arg506-->Gln mutation. In the second family 10 subjects were identified to be heterozygous for the factor V Arg506-->Gln mutation; among them 2 developed a thrombotic event. In the same family 2 individuals were found to be homozygous for the mutation: the first had a myocardial infarction at age 25 yr and the second suffered from multiple episodes of deep venous thrombosis and had a stroke at age 24 yr. These data show that the risk of developing deep venous thrombosis for the carriers of the factor V Arg506-->Gln mutation is high in the families investigated. Furthermore our data imply that the factor V Arg506-->Gln mutation in its homozygous form may relate to myocardial infarction and stroke.

Natural and acquired inhibitors of hemostasis in selected symptomatic outpatients with venous thromboembolic disease.

BACKGROUND AND OBJECTIVE: Deficiencies of natural inhibitors and the presence of lupus anticoagulant are important risk factors leading to venous thromboembolic events. Before resistance to activated protein C (APC-R) was identified, the overall prevalence of inherited abnormalities of hemostasis in non-selected outpatients with venous thromboembolic disease was under 10%. This cast doubts on the of cost effectiveness and clinical significance of assaying hemostasis inhibitors in all such patients. The goal of this study is to evaluate the prevalence of inherited and acquired abnormalities of hemostasis in younger symptomatic outpatients with objectively diagnosed venous thromboembolic disease (VTD). METHODS: From October 1994 to October 1996, we diagnosed, treated and followed 191 consecutive outpatients with an objective diagnosis of venous thromboembolic disease, and assayed natural and acquired hemostasis inhibitors in 81 of them aged less than 50; in addition, 129 relatives of patients with inherited deficiencies were evaluated. RESULTS: Twenty-six of the patients under age 50 showed inherited deficiencies of natural inhibitors (3 antithrombin, 5 protein C, 3 protein 5 and 14 APC-R, 1 dysfibrinogenemia) and 8 patients had lupus anticoagulant (LA): abnormalities of hemostasis were found in 41.9% (95% confidence interval 31.1-53.5). In older selected patients, 60% (95% confidence interval 40.6-77.3) of the subjects showed abnormalities. Seventy-two of the relatives displayed natural inhibitor deficiencies; 88.5% of the families studied had at least one relative with the same defect as the propositus. INTERPRETATION AND CONCLUSIONS: A simple selection based on age, clinical and family history shows the existence of a high prevalence and the important clinical significance of abnormalities of hemostasis in symptomatic outpatients with venous thromboembolic disease.

Catastrophic antiphospholipid syndromes in systemic lupus erythematosus.

The objective of this study was to look for the occurrence of catastrophic antiphospholipid syndromes (APS) and to try to detect discriminating factors for predicting a worse prognosis, related to Lupus anticoagulant (LA) and antiphospholipid antibodies (aPL), in systemic lupus erythematosus (SLE) with main renal involvement. Regression, recursive partition and logistic regression analyses were applied to our 80 SLE patients prospectively followed up since 1980. Immunologic and other laboratory parameters including beta 2-glycoprotein 1 dependence, resistance to activated protein C caused by a substitution on the coagulation factor V gene, induction of monocyte procoagulant activity. Regression studies demonstrated an overall worse prognosis in term of both thrombosis and death for the group of LA/aPL positive patients (33/80). However, recursive partition analysis was able to isolate a small high risk-subgroup (8/33) characterized by persistent LA/aPL antibodies positive result, widespread signs of noninflammatory vasculopathy (skin, brain, kidney) and renal pathology mimicking that of thrombotic microangiopathy or arteriolosclerosis, also in the absence of classic SLE-nephritis. Only in this subset, three catastrophic APS were recorded, while, in traditional SLE nephritis, even persistent LA/aPL positive results (sometimes after one previous thrombosis) did not seem to imply a particularly severe prognosis. All serologic criteria employed are unable to identify high-risk patients. We conclude that catastrophic APS is a rare event in renal SLE. Before more predictive serologic markers become available, a simple algorithm, dealing with clinical data and renal histologic patterns, may help physicians to identify putatively high risk-LA/aPL antibodies in SLE patients with main renal involvement. This ominous subset does not belong to the group of classic SLE-nephritis.

Absence of mutations at the activated protein C cleavage sites of factor VIII in 125 patients with venous thrombosis.

Resistance to activated protein C (APC), caused by a mutation at amino acid position Arg506 of the factor V gene, has recently been identified as the most prevalent genetic defect associated with venous thrombosis. Similarly to factor V, mutations at the cleavage sites of factor VIII for APC may occur in patients with venous thrombosis. Here we have analysed 125 consecutive patients with incidental or recurrent venous thromboembolism for the presence of mutations at the cleavage sites for APC at amino acid positions Arg336 and Arg562 of factor VIII. Our findings indicate that mutations at these amino acid positions of factor VIII do not occur in the patient group analysed.