Measurement of factor XIII (FXIII) activity by an automatic ammonia release assay using iodoacetamide blank-procedure: no more overestimation in the low activity range and better detection of severe FXIII deficiencies.

BACKGROUND: Laboratory investigation with specific factor XIII (FXIII) assays plays a crucial role in diagnosis of FXIII deficiency. According to the International Society on Thrombosis and Hemostasis (ISTH), it is necessary a blank sample with iodoacetamide, provided by the kit or locally prepared, when the ammonia release assays are used, to avoid FXIII activity overestimation. METHODS: In this study we set up a modification of the Berichrom FXIII chromogenic assay, in which iodoacetamide was added by the BCS analyzer in the reaction mixture of the blank sample, without modifications of the original reagents. We analyzed 100 plasma samples of outpatients with clinical symptoms suggestive of a bleeding diathesis (20 samples had FXIII activity <20%). RESULTS: In all samples blank subtraction significantly reduced FXIII activity, mostly in the low activity range group (from 10.1% to 2.4%, p<0.0001). In this group correction with iodoacetamide also increased the agreement with the immunoassay and allowed FXIII activity measure up to 0%. CONCLUSIONS: Despite the low number of samples included in the study, the described automatic procedure seemed to decrease FXIII activity overestimation and, especially for low activity range samples (<20%), to improve the agreement between FXIII activity and concentration. Our data suggested that iodoacetamide correction could allow the detection of severe FXIII deficiencies (activity <5%) otherwise undiagnosed using the original method.

Risk of venous thromboembolism recurrence: high negative predictive value of D-dimer performed after oral anticoagulation is stopped.

In some patients with previous venous thromboembolism (VTE) D-dimer levels (D-Dimer) tend to increase after oral anticoagulant therapy (OAT) is stopped. The aim of our study was to evaluate the predictive value of D-Dimer for the risk of VTE recurrence after OAT withdrawal. After a first episode of deep vein thrombosis (DVT) of the lower limbs and/or pulmonary embolism (PE), 396 patients (median age 67 years, 198 males) were followed from the day of OAT discontinuation for 21 months. D-dimer was measured on the day of OAT withdrawal (T1), 3-4 weeks (T2) and 3 months (+/- 10 days, T3) thereafter. The main outcome events of the study were: objectively documented recurrent DVT and/or PE. D-dimer was found to be increased in 15.5%, 40.3% and 46.2% of the patients at T1, T2 and T3, respectively. In 199 (50.2%) patients, D-dimer levels were elevated in at least one measurement. During a follow-up of 628.4 years, 40 recurrences were recorded (10.1% of patients; 6.4% patient-years of follow-up). D-dimer was increased in at least one measurement in 28 of these cases, but remained normal in 11 subjects (three of whom had recurrent events triggered by circumstantial factors, three with malignancy-associated factors) (in one subject D-dimer was not measured). The negative predictive value (NPV) of D-dimer was 95.6% (95% CI 91.6-98.1) at T3 and was even higher (96.7%; 95% CI 92.9-98.8) after exclusion of the six recurrences due to circumstantial factors. Only five idiopathic recurrences occurred in the 186 patients with consistently normal D-dimer. In conclusion, D-dimer has a high NPV for VTE recurrence when performed after OAT discontinuation.

Additive thrombin inhibition by fast moving heparin and dermatan sulfate explains the anticoagulant effect of sulodexide, a natural mixture of glycosaminoglycans.

INTRODUCTION: The aim of the study was to evaluate the mechanism of the anticoagulant action of sulodexide, a mixture of glycosaminoglycans (GAGs) composed of dermatan sulfate (DS) and fast moving heparin (FMH), in vitro. MATERIALS AND METHODS: Thrombin clotting time (TCT) was measured in human platelet poor plasma (PPP). A chromogenic substrate assay was used to determine the pseudo-first order constant kinetic of thrombin inhibition (k'=k(obs)/min) either in defibrinated PPP or antithrombin (AT) or heparin cofactor II (HCII) depleted defibrinated PPP in the absence and presence of sulodexide or its components, alone and in combination. The interaction between DS and FMH was analysed by both the algebraic fractional and isobole graphical methods. RESULTS: Sulodexide, DS and FMH produced a dose-dependent prolongation of TCT with unclottable TCT at sulodexide above 4 microg/ml and at DS or FMH above 5 microg/ml. Sulodexide and its components alone and in combination produced a dose-dependent linear increase in the rate of thrombin inhibition in defibrinated PPP. The algebraic fractional and the isobole graphical methods indicated an additive effect between DS and FMH. In AT depleted PPP, the dose-dependent increase in k' produced by sulodexide was significantly lower than in PPP, while the dose-dependent increase in k' produced by DS was similar to the increase produced in PPP. In HCII depleted PPP, the dose-dependent increase in k' produced by sulodexide was significantly lower than in PPP, while the dose-dependent increase in k' produced by FMH was similar to the increase produced in PPP. CONCLUSIONS: Thrombin inhibition produced by sulodexide is due to the additive effect of its components, namely, HCII catalysis by DS and AT catalysis by FMH.