Alessandra Biasiolo

Reversal of excessive effect of regular anticoagulation: low oral dose of phytonadione (vitamin K1) compared with warfarin discontinuation.

To determine the best way to reverse the excessive effect of regular anticoagulation in patients with INR > 5 and no bleeding complications, 23 patients with INR > 5 were randomly subdivided into two groups: group A (n = 12) discontinued warfarin for one day and group B (n = 11) received 2 mg of vitamin K1 orally in addition to the usual warfarin dose. INR was determined after 24 h (day 1), after which both groups continued with their usual dose of warfarin. After 48 h (day 2), warfarin dosage was changed according to the INR value. On day 9, INR values were determined again. Five out of twelve patients in group A had INR values > 5 on day 1. One patient in group A had an INR value < 5 both on days 1 and 2. All eleven patients in group B had INR values < 5 on day 1, and all but one on day 2. On day 9, INR values were acceptable (INR 2.0-4.5) in ten group A patients and eight group B patients. These findings suggest that a low oral dose of vitamin K1 is a convenient treatment for excessive anticoagulation in patients with no bleeding complications.

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

Summary.  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‐β2‐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.

A two‐step coagulation test to identify antiβ2‐glycoprotein I lupus anticoagulants

Summary.  Lupus anticoagulants (LA) are immunoglobulins which inhibit phospholipid (PL)‐dependent coagulation tests. LA are not specific, as they may reflect the presence of antibodies to human prothrombin, human β2‐Glycoprotein I (β2GPI), an association of previous antibodies or other antibodies. Antibodies to human β2GPI act as in vitro anticoagulants by enhancing the binding of β2GPI to PL, and this binding may be influenced by calcium ion concentration. A reduction in final calcium concentration, from 10 mm to 5 mm, increased coagulation times in both dilute Russell Viper Venom Time (dRVVT) and dilute Prothrombin Time (dPT) when plasmas of patients with antiβ2GPI antibodies were used. Ten LA patients showed increased dRVVT and dPT ratios from means of 1.5 to 1.7 (P < 0.001) and 2.4 to 4.3 (P = 0.002), respectively. Instead, all LA‐positive antiβ2GPI antibody‐negative patients showed decreased coagulation times from mean ratios of 1.5 to 1.3 (P = 0.004) in dRVVT and from 2.0 to 1.5 (P = 0.01) in dPT. These results are confirmed by running dRVVT of normal plasma spiked with affinity purified IgG antiβ2GPI antibodies. Therefore, when a PL–dependent coagulation test is run twice, at different final calcium concentrations, antiβ2GPI LA can be identified.

Lupus Anticoagulant (LA) Testing: Performance of Clinical Laboratories Assessed by a National Survey Using Lyophilized Affinity-purified Immunoglobulin with LA Activity

BACKGROUND Lupus anticoagulant (LA) screens are frequently ordered in the workup of thrombophilic patients or women with fetal loss. The sensitivity and specificity of LA detection vary depending on the choice of tests, cutoff values, and results interpretation. This variation is detrimental to patient management because persistent LA positivity in patients with a history of thrombosis is a requisite for long-term anticoagulation therapy. Numerous surveys have been performed to assess the state of the art for LA diagnosis. The control plasmas used in these surveys were from LA-positive or -negative patients or were normal plasmas with monoclonal antibodies against human beta(2)-glycoprotein I (beta(2)-GPI) added. METHODS A large number of laboratories were asked to test a common set of lyophilized plasmas for LA, including three normal plasmas, to which increasing amounts of affinity-purified IgG from a patient positive for anti-beta(2)-GPI had been added, and three LA-negative plasmas: one normal, one with a coagulation deficiency, and one with heparin. RESULTS Overall, 69, 68, and 59 of 70 participants were able to detect LA in plasmas with high, intermediate, and low potency (sensitivity, 99%, 97%, and 84%). Conversely, 69, 50, and 53 of 70 were able to rule out LA in the normal, heparinized, and deficient plasma (specificity, 99%, 71%, and 76%). CONCLUSIONS Sensitivity for LA detection is satisfactory, whereas specificity could be improved. Surveys for LA detection should be carried out on a regular basis because they may help improve performance. Plasmas containing graded amounts of affinity-purified human anti-beta(2)-GPI may be used as a convenient source of well-characterized naturally occurring LA material.

Accuracy of a portable prothrombin time monitor (Coagucheck) in patients on chronic oral anticoagulant therapy: a prospective multicenter study.

A portable prothrombin time (PT) monitor allows patients on oral anticoagulant therapy (OAT) to measure their PT at home. The purpose of the study was to evaluate the accuracy and precision of a portable PT monitor (Coagucheck, Roche Diagnostics, Mannheim, Germany) as compared with laboratory methods. The prospective study was conducted in four centers of the Italian Federation of Anticoagulation Clinics. A one-month instruction phase was followed by a six-month surveillance phase. Seventy-eight subjects on stable OAT (48 men, 30 women, age range: 18-75) were selected on a volunteer basis. Dual measurements of INR values were performed in each subject both from finger capillary blood by the monitor and from venous blood by the Anticoagulation Clinic laboratory in three instruction sessions. The mean difference (bias) of the monitor INR results when compared with the average of laboratory INR and monitor INR results was -0.025 (limits of agreement-LA: -0.84/+0.81 INR units). The mean bias was -0.0675 (LA: -0.37/+0.23), +0.018 (LA: -0.39/+0.35), and +0.039 (LA: -0.49/+0.55), respectively, for INR values lower than 2.0, between 2.0 and 3.0, and greater than 3.0. The overall precision coefficient of monitor INR was 0.370, while it was 0.23, 0.46, 0.29, and 0.21, respectively, in Centers 1, 2, 3, and 4. The overall variation coefficient was 6.5% while it was 3.7%, 8.5%, 4.7%, and 4.9%, respectively, in Centers 1, 2, 3, and 4. Coagucheck has an acceptable level of accuracy for INR values in the range between 2.0 and 3.0. A wide variation in monitor performance was found among centers.

A Comparison of Lupus Anticoagulant–Positive Patients With Clinical Picture of Antiphospholipid Syndrome and Those Without

Among antiphospholipid antibodies, Lupus Anticoagulant (LAC) is recognized as the strongest risk factor for thromboembolic events or pregnancy morbidity.1 The presence of LAC in a subject with previously documented thromboembolism or a significant history of pregnancy loss defines the Antiphospholipid Syndrome (APS). Some patients, however, are diagnosed with LAC without ever having experienced previous vascular thrombosis or pregnancy morbidity. The antiphospholipid antibody profiles of LAC positive patients with or without associated clinical features of APS have been evaluated by us in a multicenter study. Centers affiliated with Italian Federation of Thrombosis Centers (FCSA) were invited to identify consecutive LAC positive patients diagnosed over a 1-year period. Three hundred twenty-one recruited patients were contacted and after giving informed consent they underwent testing for LAC after at least 12 weeks from the first one using routine laboratory procedures. LAC was not confirmed by Thrombosis Centers in 19 patients (6 were …

SERPINB3 modulates TGF-beta expression in chronic liver disease.

Transforming growth factor-β1 (TGF-β1) is the master cytokine in the pathogenesis of liver fibrosis. TGF-β1 and extent of fibrosis were correlated recently to the serpin SERPINB3 in idiopathic pulmonary fibrosis, a chronic disease recalling liver cirrhosis. The aim of this study was to assess the relation between SERPINB3, TGF-β1 and fibrosis in chronic liver diseases and to determine the effect of this serpin on TGF-β1 expression using in vitro models. SERPINB3 and TGF-β1 were evaluated in liver biopsies of 94 patients with chronic liver disease. The effect of SERPINB3 on TGF-β1 expression was determined in primary human hepatocytes, HepG2 and Huh7 cells transfected with intact SERPINB3 human gene or with reactive site loop deleted mutants. A significant correlation between TGF-β1 and SERPINB3 at the protein level was observed in liver biopsies, confirmed by a positive correlation at mRNA level. Both proteins were correlated to the extent of liver fibrosis. All transfected cells showed increased TGF-β1 mRNA and protein production and the integrity of the reactive site loop of the serpin was crucial to achieve this effect. In conclusion, chronically damaged hepatocytes produce SERPINB3 and TGF-β, and the anti-protease activity of this serpin might be implicated in TGF-β1 induction.

Monitoring SCCA-IgM complexes in serum predicts liver disease progression in patients with chronic hepatitis.

Summary.  About 30% of the patients with chronic hepatitis develop a progressive liver disease and one of the most intriguing issues is the detection of noninvasive markers for fibrosis stage and disease progression. High levels of squamous cell carcinoma antigen (SCCA)‐immunoglobulin M (IgM) are detectable in hepatocellular carcinoma and their increase in cirrhotic patients can predict tumour development. As SCCA‐IgM can also be detectable at low percentages in patients with chronic hepatitis, the aim of this study was to assess SCCA‐IgM complexes in relation to disease outcome in this group of patients. An ELISA assay was used to determine the presence of SCCA‐IgM in 188 patients with chronic hepatitis and in 100 controls. An additional serum sample was available after a median period of 6 years in 57 untreated patients: these patients were subdivided in group A, including eight patients with a fibrosis score increase ≥2 in a second liver biopsy and group B, including 49 patients without fibrosis progression during a similar follow up. SCCA‐IgM complexes were detectable in 63 of 188 (33%) patients but in none of the controls. A significant increase of SCCA‐IgM levels over time was observed in patients with fibrosis progression (mean ± SD: 117 ± 200 U/mL/year), but not in those without histologic deterioration (mean ± SD: –8.8 ± 31 U/mL/year, P < 0.0001). In conclusion, monitoring SCCA‐IgM levels over time appears a useful approach to identify patients with chronic hepatitis at higher risk for cirrhosis development.

A comparison between six- and four-week intervals in surveillance of oral anticoagulant treatment

We determined whether international normalized ratio (INR) monitoring at 6 weeks rather than 4 weeks would benefit patients and reduce costs. Patients receiving stable oral anticoagulation treatment (target INR, 3.0) with a prosthetic mechanical heart valve for more than 6 months were randomized for a maximum interval between INR determinations of 6 weeks (group 1, n = 59) or 4 weeks (group 2 [control], n = 65). Patients were followed up for 2 years. The primary end point of the study was the biologic risk of overanticoagulation or underanticoagulation, estimated as the rate of values at risk (INR, < 1.5 and > 5). The rates of INR values at risk for hemorrhagic (INR, > 5) or thromboembolic (INR, < 1.5) complications were 3.27% in group 1 and 3.09% in group 2 (P = .81). The INRs of patients in group 1 trended toward higher values, but no difference between groups was observed in time spent at various INR ranges by using the method of linear change. The mean time between INR determinations was 24.9 +/- 18.1 days (1.20 per month) in group 1 and 22.5 +/- 9.5 days (1.33 per month) in group 2 (P < .0003). For patients in stable condition with a prosthetic heart valve who are monitored at an anticoagulation clinic, a 6-week interval between INR determinations does not increase the biologic risk of thromboembolic or hemorrhagic events.

SERPINB3 is associated with TGF-β1 and cytoplasmic β-catenin expression in hepatocellular carcinomas with poor prognosis.

Background:Hepatocellular carcinoma (HCC) is one of the most important sanitary problems for its prevalence and poor prognosis. To date, no information is available on the prognostic value of the ov-serpin SERPINB3, detected in primary liver cancer but not in normal liver. The aim of the study was to analyse SERPINB3 expression in liver cancer in relation with molecular signatures of poor prognosis and with clinical outcome.Methods:Liver tumours of 97 patients were analysed in parallel for SERPINB3, TGF-β and β-catenin. In a subgroup of 67 patients with adequate clinical follow-up, the correlation of molecular findings with clinical outcome was also carried out.Results:High SERPINB3 levels were detectable in 22% of the patients. A significant correlation of this serpin with TGF-β at transcription and protein level was observed, whereas for β-catenin a strong correlation was found only at post-transcription level. These findings were in agreement with transcriptome data meta-analysis, showing accumulation of SERPINB3 in the poor-prognosis subclass (S1). High levels of this serpin were significantly associated with early tumour recurrence and high SERPINB3 was the only variable significantly associated with time to recurrence at multivariate analysis.Conclusions:SERPINB3 is overexpressed in the subset of the most aggressive HCCs.