摘要:

With the identification of common single locus point mutations as risk factors for thrombophilia, many DNA testing methodologies have been described for detecting these variations. Traditionally, functional or immunological testing methods have been used to investigate quantitative anticoagulant deficiencies. However, with the emergence of the genetic variations, factor V Leiden, prothrombin 20210 and, to a lesser extent, the methylene tetrahydrofolate reductase (MTHFR677) and factor V HR2 haplotype, traditional testing methodologies have proved to be less useful and instead DNA technology is more commonly employed in diagnostics. This review considers many of the DNA techniques that have proved to be useful in the detection of common genetic variants that predispose to thrombophilia. Techniques involving gel analysis are used to detect the presence or absence of restriction sites, electrophoretic mobility shifts, as in single strand conformation polymorphism or denaturing gradient gel electrophoresis, and product formation in allele-specific amplification. Such techniques may be sensitive, but are unwielding and often need to be validated objectively. In order to overcome some of the limitations of gel analysis, especially when dealing with larger sample numbers, many alternative detection formats, such as closed tube systems, microplates and microarrays (minisequencing, real-time polymerase chain reaction, and oligonucleotide ligation assays) have been developed. In addition, many of the emerging technologies take advantage of colourimetric or fluorescence detection (including energy transfer) that allows qualitative and quantitative interpretation of results. With the large variety of DNA technologies available, the choice of methodology will depend on several factors including cost and the need for speed, simplicity and robustness.

ISSN:0957-5235    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

The optimal duration of oral anticoagulant therapy after a first episode of venous thromboembolism (VTE) is still a matter of debate. It is essential to balance the desired effect of the anticoagulants in reducing recurrences against the risk of major bleeding. The aims of this paper are to describe the current concepts in this field. Recent data, based on randomized controlled trials, suggest that it is necessary to tailor the duration of anticoagulation individually according to the topography of VTE and the presence of risk factors. A 6-week treatment for patients with isolated calf vein thrombosis is sufficient. For proximal thrombosis and/or pulmonary embolism, a short anticoagulant course is sufficient in patients with temporary risk factors (3 months), and a longer anticoagulant course (6 months at least) is recommended for cases with permanent risk factors or idiopathic VTE. For these high-risk of recurrence patients, an assessment of low- or fixed-dose oral anticoagulation is necessary in order to reduce the bleeding risk. It is not possible to precisely determine the optimal duration with the available data. We have already performed a meta-analysis on summary data that suggests a long course of oral anticoagulant therapy is superior to a short course. An individual meta-analytic approach is needed to draw more precise conclusions on an interesting and important clinical topic.

ISSN:0957-5235    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

The present study investigates the effect of bezafibrate on lipid levels and coagulofibrinolytic factors. Subjects enrolled in the study included 124 postmenopausal women with hypertriglyceridemia. We examined the levels of tissue plasminogen activator (t-PA), plasminogen activator inhibitor type-1 (PAI-1), and blood coagulation factors VII and X as parameters of coagulofibrinolysis. After 12 weeks of bezafibrate therapy, mean serum triglyceride (TG) and remnant-like particle lipoprotein cholesterol (RLP-C) levels significantly decreased from baseline. Activated factor X levels decreased significantly by 11.3% (P< 0.01) and the ratio of tPA/PAI-1 (0.146 ± 0.07) increased significantly by 37.7% from baseline (P< 0.05). The rates of change in activated factor VII and PAI-1 showed a significant positive correlate with the rates of change in TG and RLP-C (P= 0.001). These present findings suggest that bezafibrate improves triglyceride-rich lipoprotein metabolism and coagulofibrinolytic activity by increasing fibrinolysis in postmenopausal women with hypertriglyceridemia.

ISSN:0957-5235    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

D-Dimer testing has been suggested as a non-invasive method for the exclusion of pulmonary embolism (PE) and deep vein thrombosis (DVT). In this study, we compared a new method, the Miniquant D-dimer (Biopool International, Ventura, California, USA) to other previously validated D-dimer methods used for the purpose. Patients who were undergoing a definitive diagnostic study for thromboembolism had a blood sample drawn at that time. A whole-blood D-dimer (SimpliRed; Agen Biomedical Ltd, Brisbane, Australia) test was performed, and residual plasma was frozen and later analyzed using two enzyme-linked immunosorbent assay (ELISA) methods (D-dimer Gold; Agen, and Asserachrome D-Di; Stago International, Parsippany, New Jersey, USA) and the Miniquant D-dimer. Once all samples were analyzed, the correlation and accuracy of the Miniquant was compared with the ELISA method using Spearman's regression and Dunn's multiple paired comparison. All D-dimer methods were compared with radiographic studies. The data was analyzed collectively and segregated into in-patient (n= 112) and out-patient (n= 143) populations. The Miniquant D-dimer sensitivity, specificity and negative predictive value (NPV) for all patients were 95, 21, and 94% for DVT, and 100, 26, and 100% for PE. This new D-dimer method demonstrates acceptable sensitivity in patients with PE and DVT and, based on the high NPV of this method, it can be used for the exclusion of thromboembolism.

ISSN:0957-5235    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Platelet volume has been reported to be increased in vascular disease. Therefore, we studied the relationship of mean platelet volume and platelet count as well as flow cytometrically measured platelet size and platelet function in 50 patients with peripheral arterial disease and 50 healthy volunteers. Platelet activation was measured by P-selectin expression analysis on resting and on stimulated platelets, and the determination of platelet aggregates and platelet-derived microparticles using flow cytometry. P-Selectin expression on platelets was significantly elevated in patients suffering from peripheral arterial disease (allP< 0.0001). Platelet aggregates (P< 0.0001) and platelet-derived microparticles (P< 0.0001) were significantly higher in the patient group compared with controls, whereas mean platelet volume and platelet count showed no significant differences. Platelet count was inversely related to mean platelet volume in patients and controls (r= −0.43,P< 0.001). The present study supports the hypothesis of platelet hyperreactivity and circulating activated platelets in peripheral arterial disease. Mean platelet volume, and platelet count cannot be used as predictive markers for platelet activation in peripheral arterial disease patients.

ISSN:0957-5235    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

This study reports an evaluation of current laboratory practice for the diagnosis of von Willebrand's disorder (VWD) by means of a multi-laboratory (n= 19) survey (the ‘Second Australasian VWD Survey'). Results are compared with a previously conducted but similarly comprehensive survey (`Survey-1'). Samples comprised a new set of seven plasmas: (i) Type 3 VWD; (ii) Type 2B VWD; (iii) Moderate Type 1 VWD/Haemophilia A combined defect; (iv) Normal individual; (v) Mild Type 1 VWD; (vi) Type 2M/2A VWD; (vii) Type 2N VWD. Overall, many current findings confirmed those reported in Survey-1 [including between-method analysis, within-method analysis, inter-laboratory assay variation, sensitivity to low levels of von Willebrand Factor (vWF), detection of functional vWF ‘discordance', and appropriateness of diagnostic predictions]. Novel findings include: (i) although vWF:collagen binding activity (vWF:CBA) performed better than vWF:ristocetin cofactor (vWF:RCof) assay in identification of functional discordance in Type 2B VWD, both assays performed equally in identification of discordance in the Type 2M/2A VWD; (ii) most laboratories failed to identify the Type 2N VWD as a potential 2N utilizing vWF antigen (vWF:Ag) and factor VIII coagulant (FVIII:C) testing as a screening process; (iii) this particular survey was followed up by a dry workshop attended by over 45 scientists from Australia and New Zealand, and comprising representatives from most survey participants. Discussion covered many topics including the effect of blood group, the role (if any) of the bleeding time, the role of the PFA-100TM, confirmatory and additional tests, and the possibility of restricting testing to specialized centres. Consensus was reached on the following points: (i) diagnosis of VWD requires both clinical and laboratory assessment; (ii) testing should comprise FVIII:C, vWF:Ag and either/or both vWF:RCof and vWF:CBA; (iii) laboratory results should be reviewed in the light of clinical findings; and (iv) confirmatory repeat testing should be performed on a sample taken 6 weeks later.

ISSN:0957-5235    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

The coagulant activity of blood coagulation factor VII (FVII:C) can be lowered by changes in lifestyle and by therapeutic intervention, e.g. heparin infusion. The question is, however, whether FVII:C determinedex vivois a valid measure of the FVII activityin vivo. We measured plasma FVII:C, activated FVII (FVIIa), FVII protein (FVII:Ag), tissue factor pathway inhibitor (TFPI), triglycerides, and free fatty acids (FFA) before and 15 min after infusion of a bolus of unfractionated heparin (50 IU/kg body weight) in 12 healthy subjects. Additionally, we conductedin vitroexperiments to investigate the effect of unfractionated heparin and TFPI, which is released from the endothelium by heparin, on FVII:C, FVIIa, and FVII:Ag. Heparin infusion decreased triglycerides and increased FFA and TFPI. This was accompanied by significant reductions in FVIIa, FVII:C and FVII:Ag.In vitro, anti-TFPI antibodies increased FVIIa and FVII:C, and heparin reduced FVIIa. The heparinase Hepzyme was unable to abolish the effect of heparin. There were noin vitroeffects on FVII:Ag. We conclude that, due to interference by TFPI and heparin in post-heparin plasma, it is impossible to measure thein vivoFVII activity by means of FVII clotting assays. These assays should therefore not be used to measure the coagulation status of patients in heparin therapy, unless extraordinary precautions are taken to eliminate TFPI and heparin effectsex vivo. The observed effect of heparin on FVII:Ag should be investigated further.

ISSN:0957-5235    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Different assays for the assessment of protein S (PS) functional activity are commercially available. We were able to show that, considering the influence of factors known in respect of PS, good agreement can be reached between the results of the determination of free PS as obtained using an immunoassay with monoclonal antibodies and the determination of PS activity as obtained using a test based on activated factor X (factor Xa). However, values of PS activity higher than free PS concentration were obtained in plasma samples taken from patients undergoing therapy with low molecular weight (LMW) heparin. Anin vitroincubation of plasma samples with LMW heparin in varying concentrations led, in every case, to an increase of clotting times and thus to an increase of PS activity. In all investigations, the ratios of clotting time with heparin to that without heparin were higher in plasma samples containing PS than in PS-deficient plasma. This result was independent of the use of commercially deficient plasma or the blocking of PS in reference plasma by addition of polyclonal PS antibodies. Obviously, heparin blockers in commercially available assays only neutralize the effect of conventional heparin, and the prolongation of the clotting time is mainly caused by the inhibition of factor Xa by LMW heparin. The reason for the stronger effect in plasma containing PS than in the same plasma after the blocking of PS with polyclonal antibodies as well as in PS-deficient plasma is unclear. Due to the unrecognizable influence of LMW heparin on global clotting assays, the assessment of PS activity values without clear documentation of the application of LMW heparin can lead to improper diagnoses.

ISSN:0957-5235    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Thirty-eight obese children and adolescents were investigated for a possible relation between cholesterol and markers of platelet activation, endothelial cell dysfunction, and activation of the coagulation system. Soluble P-selectin, von Willebrand factor antigen (vWf-Ag), D-dimer, and prothrombin fragment 1 + 2 (F1 + 2) were determined by enzyme-linked immunosorbent assays, and factor VIII coagulant activity (VIIIc) was measured by means of one-stage clotting assay. Cholesterol correlated significantly with log P-selectin (r= 0.43,P= 0.003) and log D-dimer (r= 0.33,P= 0.02). Cholesterol did not correlate with vWf-Ag, factor VIIIc, and F1 + 2. Log P-selectin correlated significantly with log D-dimer (r= 0.42,P= 0.003), which remained significant after adjustment for cholesterol (P= 0.02). Log D-dimer correlated significantly with F1 + 2 (r= 0.38,P= 0.01). Our study demonstrates that, in obese children and adolescents, cholesterol is significantly associated with P-selectin and D-dimer, and suggests an unfavorable intercorrelation between metabolic and hemostatic risk factors for coronary heart disease in childhood obesity.

ISSN:0957-5235    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Variability of thrombotic disease among individuals homozygous for factor V Leiden has been described. It has been shown that some thrombotic patients carry an additional genetic risk factor such as protein C, protein S, antithrombin deficiency or the G20210A mutation on the prothrombin gene. The occurrence of a deep vein thrombosis during enoxaparin prophylactic treatment in a pregnant woman homozygous for factor V Leiden, without other known prothrombotic genetic factors, led us to investigate her thrombomodulin gene. We found that the patient was heterozygous for the previously described G127← A mutation, which results in an Ala25← Thr substitution. Furthermore, for this patient, the allelic combination at the 1418 polymorphic site was C/T, which predicts an Ala455← Val replacement. Although larger studies are required, this case report suggests that thrombomodulin gene mutations could be an additional genetic risk factor for thrombosis in carriers of the factor V Leiden mutation.

ISSN:0957-5235    

出版商:OVID    

年代:2000

数据来源: OVID