Angela Rogolino

Cardiovascular and thrombophilic risk factors for idiopathic sudden sensorineural hearing loss.

Summary.  Background: In recent years there has been a significant increase in the diagnosis of sudden sensorineural hearing loss (SSHL) in western, countries with an incidence of 20 of 100 000 people affected every year. No clear causes for this disease have been found thus far, but cochlear ischemia has been hypothesized in patients in whom an infectious episode or acoustic neurinoma have been excluded. Objectives: The aim of this case–control study was to investigate a number of acquired and inherited thrombophilic risk factors [antithrombin, protein C and S; factor V (FV) Leiden, FII polymorphism; lupus anticoagulant (LA); anticardiolipin (aCL) antibodies; fasting homocysteine (Hcy); lipoprotein(a) (Lp(a)); plasminogen activator inhibitor‐1 (PAI‐1)] in addition to cardiovascular risk factors in patients with idiopathic SSHL (ISSHL). Patients and methods: We investigated 155 patients (67 male/88 female; age: 55 (range 19–79 years) with a diagnosis of ISSHL within 30 days from the onset of symptoms, and 155 controls (67 male/88 female; age 54 (range 19–78 years). Fasting Hcy levels were significantly higher in patients than in controls [11.6 (6.7–60) μmol/L vs. 8.7 (5.0–24) μmol/L] as well as PAI‐1 levels [19 (2–95) mg/dL vs. 14.5 (4.0–87) mg/dL]. Lupus anticoagulant was present in 13 of 155 (8.4%) patients; 20 patients (12.9%) had positivity of aCL (four IgM and 16 IgG). In no patient was a deficiency of physiological clotting inhibitors antithrombin, protein C and protein S found. No significant differences between patients and controls were observed for Lp(a) plasma levels [111 (1–1146) mg/L vs. 103 (11–695) mg/L] and for the presence of FV Leiden (4.5% vs. 4.5%) and FII variant G20210A (3.8% vs. 3.2%). Results and conclusions: Independent risk factors for ISSHL at the multivariate analysis (adjusted for age, sex and the traditional cardiovascular risk factors) were the positivity of aCL: OR 5.6 (95% CI 2.0–15.3); cholesterol levels within the second and third tertiles (with respect to the first tertile): T2 = OR 4.8 (95% CI 1.9–12.6)/T3 = OR 19 (95% CI 7–50.1); PAI‐1 and Hcy levels within the third tertile (with respect to the first tertile): OR 20 (95% CI 7.8–78) and OR 4.0 (95% CI 2.0–8.1), respectively. These preliminary data suggest that hypercholesterolemia, hyperhomocysteinemia, elevated PAI‐1 levels and anticardiolipin antibodies are associated with ISSHL, so indirectly supporting the hypothesis of a vascular occlusion in the pathogenesis of the disease.

Hypercoagulability, high tissue factor and low tissue factor pathway inhibitor levels in severe ovarian hyperstimulation syndrome: possible association with clinical outcome

&NA; During ovarian gonadotrophin stimulation for ovulation induction or in vitro fertilization, a clinical severe ovarian hyperstimulation syndrome (OHSS) may occur. Only few studies have investigated the mechanism responsible for the alterations of the haemostatic system in women affected by severe OHSS. The aim of the present study was to investigate the correlation between the magnitude of ovarian stimulation and the increase in fibrin formation and degradation in severe OHSS. Twenty‐five patients (age range 23‐43 years) who were hospitalized for severe OHSS, 25 women undergoing in vitro fertilization who did not develop OHSS (case‐control group) and 25 healthy age‐matched women (healthy control group) were investigated. On the day of admission a number of haemostatic markers, including D‐dimer, thrombin‐antithrombin complexes (TAT), prothrombin fragment 1+2 (F1 + 2), plasmin‐antiplasmin complexes (PAP), tissue factor (TF), tissue factor pathway inhibitor (TFPI) and von Willebrand factor antigen (vWF), were examined. In patients with severe OHSS, TF, D‐dimer, TAT, F1 + 2, PAP and vWF antigen plasma levels were significantly higher than those observed both in the case‐control group and in healthy controls, whereas TFPI levels were significantly lower (P < 0.005) with respect to both case‐controls and healthy controls. D‐Dimer levels were related with serum oestradiol levels and oocyte number recovered (r= 0.45, P < 0.001 and r=0.47, P < 0.001, respectively). D‐Dimer and TAT levels were significantly (P < 0.05 and P < 0.005, respectively) higher in OHSS patients with unsuccessful pregnancy outcome (D‐dimer, 226.5, 56‐1449 ng/ml; TAT, 19.8, 3.1‐82.6 μg/l) with respect to those with successful outcome of pregnancy (D‐dimer, 145, 29‐330 ng/ml; TAT, 5.0, 1.0‐19.6 μg/l). Our data indicate that a marked hypercoagulability with alterations of TF and TFPI levels is detectable in patients with severe OHSS and that it is related to the clinical outcome. Blood Coagul Fibrinolysis

Evaluation of automated immunoassays in the diagnosis of heparin induced thrombocytopenia

BACKGROUND Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies that recognize platelet factor 4/heparin (PF4/hep) complexes. The in vitro demonstration of PF4/hep antibodies using functional and immunological methods is essential for optimal management of patients suspected to have HIT. Since functional assays are technically challenging and limited to specialized laboratories, antigen-binding assays are commonly used in routine laboratories. STUDY DESIGN Blood samples from 448 consecutive patients in whom HIT was suspected were investigated using a latex agglutination test HemosIL® HIT-Ab(PF4-H) (HemosIL-Ab), two chemiluminescence tests HemosIL AcuStar HIT-Ab(PF4-H) (HemosIL AcuStar-Ab) and AcuStar HIT-IgG(PF4-H) (HemosIL AcuStar-IgG), an in-house PF4/hep IgG enzyme immunoassay (EIA) and the heparin induced platelet aggregation (HIPA) test. RESULTS Antibodies against PF4/hep were detectable in 44 out of 119 samples using HemosIL-Ab among which 20 samples were also reactive in the HIPA; and in 122, 64 and 108 out of 448 sera using HemosIL AcuStar-Ab, HemosIL AcuStar-IgG and in-house PF4/hep IgG-EIA, respectively, among which 52 sera were also reactive in the HIPA. All assays had high sensitivities of >95% for platelet activating antibodies; however, they differed in their specificities. The highest specificity and positive predictive value was observed by HemosIL AcuStar-IgG (96% and 78%, respectively). CONCLUSION Automated immunoassays are useful in the laboratory investigations of HIT and present a potential improvement toward standardization of laboratory investigations of HIT. The high positive predictive capability may justify treating the patient with alternative anticoagulants without waiting for the results of a functional assay.

Index Measured at an Intermediate Altitude to Predict Impending Acute Mountain Sickness

PURPOSE Acute mountain sickness (AMS) is a neurological disorder that may be unpredictably experienced by subjects ascending at a high altitude. The aim of the present study was to develop a predictive index, measured at an intermediate altitude, to predict the onset of AMS at a higher altitude. METHODS In the first part, 47 subjects were investigated and blood withdrawals were performed before ascent, at an intermediate altitude (3440 m), and after acute and chronic exposition to high altitude (Mount Everest Base Camp, 5400 m (MEBC1 and MEBC2)). Parameters independently associated to the Lake Louise scoring (LLS) system, including the self-reported and the clinical sections, and coefficients estimated from the model obtained through stepwise regression analysis were used to create a predictive index. The possibility of the index, measured after an overnight stay at intermediate altitude (Gnifetti hut, 3647 m), to predict AMS (defined as headache and LLS ≥ 4) at final altitude (Capanna Margherita, 4559 m), was then investigated in a prospective study performed on 44 subjects in the Italian Alps. RESULTS During the expedition to MEBC, oxygen saturation, hematocrit, day of expedition, and maximum velocity of clot formation were selected as independently associated with LLS and were included in the predictive index. In the Italian Alps, subjects with a predictive index value ≥ 5.92 at an intermediate altitude had an odds ratio of 8.1 (95% confidence limits = 1.7-38.6, sensitivity = 85%, specificity = 59%) for developing AMS within 48 h of reaching high altitude. CONCLUSION In conclusion, a predictive index combining clinical and hematological parameters measured at an intermediate step on the way to the top may provide information on impending AMS.

Tissue factor and tissue factor pathway inhibitor levels in unstable angina patients during short-term low-molecular-weight heparin administration

Summary. High tissue factor (TF), tissue factor pathway inhibitor (TFPI) levels and a hypercoagulability state have been documented in unstable angina patients. We evaluated whether short‐term enoxaparin administration (100 IU/kg b.i.d. for 3 d) reduces the high TF levels and the hypercoagulability state, and whether it influences the fibrinolytic system in 20 unstable angina patients. On d 3, we observed a significant reduction in TF levels both 1 h and 4 h after the morning injection (−25·6% and −21·7%; P < 0·001 respectively) in comparison with the base‐line levels. Both 1 and 4 h after the morning injection on the d 3, TFPI levels significantly (P < 0·001) increased (+96·4%, +96·9% respectively) with respect to the base‐line values. After enoxaparin administration, at all observation times, thrombin–antithrombin complexes and prothrombin fragment F1 + 2 levels were significantly (P < 0·001) lower with respect to base‐line levels. We observed a slight but significant increase in tissue plasminogen activator antigen levels in preinjection samples, as well as 1 h and 4 h after enoxaparin administration, in comparison with the base‐line values. This study provides evidence that low‐molecular‐weight heparin (LMWH) administration, in addition to a reduction of hypercoagulability and a mild fibrinolytic activation, is associated with decreased TF levels, so indicating a novel mechanism of interference of LMWH with the haemostatic system.

Assessment of Fibrinolytic Activity by Measuring the Lysis Time of a Tissue Factor-induced Clot: A Feasibility Evaluation

A clot lysis time assay in which a tissue factor—induced fibrin clot is lysed by exogenously added tissue plasminogen activator has been recently reported. We evaluated the feasibility of clot lysis time in a routine hemostasis laboratory, and its correlation with thrombin activatable fibrinolysis inhibitor and plasminogen activator inhibitor-1 levels and changes with aging in 185 healthy participants. Clot lysis time was assessed by monitoring changes in turbidity during clot formation and subsequent lysis using a computerized kinetic spectrophotometric microtiter plate. After preliminary experiments, 100 and 160 ng/mL tissue plasminogen activator concentrations were chosen for the study. Clot lysis time was calculated by a new mathematical analysis of the lysis curve based on discrete derivative. Clot lysis time, thrombin activatable fibrinolysis inhibitor, and plasminogen activator inhibitor-1 plasma levels showed a normal distribution. For both concentrations of tissue plasminogen activator, clot lysis time progressively increased with increase in age (P < .0001) and was significantly correlated with thrombin activatable fibrinolysis inhibitor antigen, thrombin activatable fibrinolysis inhibitor activity, and plasminogen activator inhibitor-1 antigen (at least P < .01). During linear regression analysis, thrombin activatable fibrinolysis inhibitor and plasminogen activator inhibitor-1 antigen were found to significantly influence clot lysis time (at least P < .01). Clot lysis time determination has a good laboratory performance. Our new method of calculation is independent of the time of reading and allows a more accurate and consistent detection of both short and prolonged lysis times. Our data suggest the feasibility of the use of this test in the work of routine hemostasis laboratory.

A hypercoagulable and hypofibrinolytic state is detectable by global methods in patients with retinal vein occlusion

The pathogenesis of retinal vein occlusion (RVO), has not been well understood. Recent data have shown the efficacy of an anticoagulant therapy with LMWHs in the treatment of acute RVO suggesting the presence of a hypercoagulable state in these patients. New global tests for detection of hypercoagulability and hypofibrinolysis have become available and their application might improve the knowledge of the pathophysiology of RVO and, potentially, its treatment. The aim of our study was to evaluate coagulation and fibrinolytic alterations by two global tests in RVO patients: Endogenous Thrombin Potential (ETP) and Clot Lysis Time (CLT), respectively. We studied 81 RVO patients (40 males; median age 61 years) and a control group matched for age and sex. The ETP was measured by functional chromogenic assay and expressed as the time until thrombin burst (LagTime), Time to peak (T(max)), Peak amount of thrombin generation (C(max)) and ETP. CLT was determined by a plasma-based, tissue factor-induced clot lysis assay. C(max), ETP and CLT values were significantly higher in RVO patients than in controls (C(max)p = 0.010; ETP p < 0.001; CLT p < 0.001) and remained significantly associated with the disease at the multivariate analysis adjusted for cardiovascular risk factors. Our results indicate that -beyond the assay of different parameters associated with clotting activation and lysis- global methods might allow us to easily detect the presence of hypercoagulability and hypofibrinolysis in RVO patients. Further studies should assess the possible clinical value of our data in the management of RVO patients.

Risk factors for cardiovascular disease in renal transplant recipients: new insights

Abstract Long‐term survival of renal transplant recipients appears to be influenced by the occurrence of thromboembolic complications and cardiovascular disease. In order to investigate the prevalence of new hemostasis‐related risk factors for venous and arterial thrombosis, we investigated 63 renal transplant recipients and 66 age‐ and sex‐matched control subjects. We assayed antiphospholipid antibodies [lupus anticoagulant (LA) and anticardiolipin antibodies (aCL)], lipoprotein (a) [Lp(a)], plasminogen activator inhibitor‐1 (PAI‐1), and total homocysteine (tHcy) levels. We found a significantly higher prevalence of positivity for LA (P < 0.001); no difference was detected in the prevalence of aCL between patients and controls. PAI‐1 levels were significantly higher in renal transplant recipients than in controls [12.3 IU/ml (2‐45.5) vs 7.9 IU/ml (4‐18.0); P < 0.0001] with an odd ratio (OR) of 11.8 (4.9‐28.5) in univariate analysis and of 5.8 (2.1‐15.4) in multivariate analysis. Lp(a) levels were higher in patients then in controls [159 mg/l (1‐992) vs 100.5 mg/l (10‐412); P < 0.005] with an OR of 5.9 (1.9‐18.4) in univariate analysis and of 3.5 (0.9‐13.4) in multivariate analysis. Fasting levels of tHcy were significantly higher in renal transplant recipients [7.0 μmol/l (4.0‐68) vs 8.1 μmol/l (2.0‐24.0); P < 0.00001] with an OR of 40.4 (14.7‐111) in univariate analysis and of 33.1 (11.1‐115.5) in multivariate analysis. After methionine loading test, we documented levels of tHcy above the 90th percentile of controls in 60/63 patients (95 %). Finally, we found a significant correlation between tHcy and PAI‐1 plasma levels (r = 0.76; P < 0.000001). Our results show a high prevalence of hemostasis‐related risk factors for arterial and venous thrombosis in renal transplant recipients, suggesting the need for the investigation of these patients for the presence of these risk factors in order to improve their long‐term survival and to tailor therapy.

Lower Homocysteine Levels in Renal Transplant Recipients Treated With Everolimus: A Possible Link With A Decreased Cardiovascular Risk?

Cardiovascular disease (CVD) is the main cause of morbidity and mortality in renal transplant recipients. The incidence of CVD in this setting is approximately 5-fold greater than in age- and and gender-matched subjects. This excess cardiovascular risk is not completely explained by traditional cardiac risk factors. It has been well documented that these patients show greatly increased prevalence of both fasting and postmethionine-loading hyperhomocysteinemia (hHcy) compared with the general population. An immunosuppressive therapy based on everolimus has been demonstrated to reduce the incidence major adverse coronary events at 4 years compared with azathioprine among heart transplant recipients. In contrast, scarce data are available on the impact of everolimus on emerging risk factors, such as homocysteine (Hcy), in renal transplant recipients. The aim of this study was to evaluate the possible impact of everolimus compared with other immunosuppressive regimes among 132 stable recipients, including 91 men and 41 women who were at least 1 year after transplant with stable renal function and no clinical evidence of acute or chronic renal graft rejections. We compared 31 subjects on everolimus immunosuppressive therapy (group A) versus 101 on immunosuppressive therapy based on cyclosporine, steroids, and mycophenolate. The Hcy levels were significantly lower among group A patients compared with group B: 16.5 +/- 5 micromol/L vs 21.2 +/- 11 micromol/L; P < .005. Hyper-Hcy, defined as Hcy levels >15 micromol/L, was diagnosed in 18 out of 31 patients (51%) of group A and in 82 out of 101 patients (81%) of group B. This preliminary study demonstrates a favorable impact of everolimus on a marker of atherothrombosis which is associated with a worse vascular prognosis.

Circulating Biomarkers in Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy Patients

BACKGROUND Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited cerebral microangiopathy presenting with variable features, including migraine, psychiatric disorders, stroke, and cognitive decline and variable disability. On neuroimaging, CADASIL is characterized by leukoencephalopathy, multiple lacunar infarcts, and microbleeds. Previous studies suggest a possible role of endothelial impairment in the pathogenesis of the disease. METHODS We assessed plasma levels of von Willebrand factor (vWF) and thrombomodulin (TM) and the blood levels of endothelial progenitor cells (EPCs) and circulating progenitor cells (CPCs) in 49 CADASIL patients and 49 age-matched controls and their association with clinical/functional and neuroimaging features. RESULTS In multivariate analysis, CADASIL patients had significantly higher vWF and lower EPC levels. TM levels were similar in the 2 groups. CADASIL patients with a more severe clinical phenotype (history of stroke or dementia) presented lower CPC levels in comparison with patients with a milder phenotype. On correlation analysis, lower CPC levels were associated with worse performances on neuropsychological, motor and functional tests, and with higher lesion load on brain magnetic resonance imaging (degree of leukoencephalopathy and number of lacunar infarcts). CONCLUSIONS This is the first CADASIL series in which multiple circulating biomarkers have been studied. Our findings support previous studies on the presence and the possible modulating effect of endothelial impairment in the disease. Furthermore, our research data suggest that blood CPCs may be markers of disease severity.