Neonatal haemostasis and the management of neonatal thrombosis

Two detailed reviews of the management of neonatal thrombosis were published in 2012; one was an up‐dated version of guidance first issued in 2004 and the other was a comprehensive review. Both of these publications gave very similar advice regarding the practical aspects of the indications, dosage and management of antithrombotic therapy. The authors stated that the evidence supporting most of their recommendations for anti‐thrombotic therapy in neonates remained weak and so the therapy for a neonate with a thrombosis has to be based on an individualized assessment of estimated risk versus potential benefit. The aim of this present review is to give the treating physician an outline of the unique physiology of neonatal coagulation and how this affects the monitoring, dosing and even the choice of therapeutic strategy for the management of thrombosis in the neonate.     

The application of molecular biology techniques to haemostasis and thrombosis.

One may no longer consider coagulation research without encompassing the wealth of knowledge available from the application of molecular biology techniques. This innovative form of research has not proceeded to the exclusion of more conventional techniques, and moreover has provided support for basic scientific hypotheses. This paper discusses the molecular biology techniques currently used in the study of haemostatic and thrombotic disorders and gives an outline of the evolution of such techniques which have taken place at a staggering pace. The article includes specific examples of how results from molecular studies are directly applied to hereditary haemostatic and thrombotic diseases to support clinical diagnosis and to increase our understanding of the biochemical processes involved in the disorders. The reader is referred to several reviews for detailed lists of genetic defects revealed by molecular biological techniques.     

The pivotal role of the endothelium in haemostasis and thrombosis

The endothelium plays a pivotal role in the regulation of the haemostatic balance. The function of endothelial cells exceeds far beyond providing a non-thrombogenic inner layer of the vascular wall which maintains the blood fluidity. In physiological circumstances endothelial cells carefully prevent thrombosis by different anticoagulant and antiplatelet mechanisms. Endothelial cells are involved in all major haemostatic pathways upon vascular injury and limit clot formation to the areas where haemostasis is needed to restore vascular integrity. Failure of this complex balance between pro- and anticoagulant systems because of genetic or acquired disturbances may result in bleeding or thrombosis. Endothelial heterogeneity assures adequate homeostasis in the different organs and parts of the vascular tree. The local environment induces heterogeneous endothelial cell phenotypes determined by local needs. This heterogeneity also explains the diverse pathological responses upon disturbed vascular integrity. Localised manifestation of thrombosis in spite of systemic procoagulant disturbances depends on vascular bed-specific properties. Endothelial dysfunction not only precedes atherogenesis but may also predispose to arterial thrombosis. The potential role of the endothelium in venous thrombosis with and without overt vessel wall injury is discussed. The vast majority of endothelial cells are located in the microvessels. Therefore, it is no surprise that endothelial cells play a key role in microcirculatory diseases such as thrombotic microangiopathies and diffuse intravascular coagulation. Microcirculatory endothelial cell activation is an important feature in all thrombotic microangiopathies. In diffuse intravascular coagulation, the endothelium is the interface between inflammation and inappropriate activation of the coagulation system.     

Clinical diagnosis of overtraining using blood tests: current knowledge

PURPOSE: Overtraining results from an imbalance between training load-induced fatigue and organism's recovery abilities. Its etiology is complex and to date there is no useful clinical diagnostic tool. The purpose of this review is to discuss the blood chemistry parameters potentially useful for diagnosing overtraining in athletes. CURRENT KNOWLEDGE AND KEY POINTS: Chronic alterations of the myocyte structure may cause high plasma concentration increases of myoglobin, troponin I and creatine kinase enzyme, resulting in chemical and/or mechanical aggression. Monitoring reactive oxygen species' activity appears to be a good tool for evaluation of the metabolic stress level experienced by skeletal muscles. In energetic metabolism, a succession of chronic glycogen depletions might change the use of amino acids and lipids, inducing transient but severe hypoglycemia during exercise. A higher oxidation of circulating glutamine might cause immunosuppression (lower reactivity to inflammations and cellular traumatisms), inhibiting alarm signals during acute training. A higher branched-chain amino acid oxidation might favor free tryptophan's entry into the cerebral area, enhancing serotonin synthesis. As a consequence, asthenia and a loss of sensitivity to muscular and tendon traumatism might appear. Exercise anemia might also be a worsening factor of the physiological situation of the tired athlete, inducing predisposition to overtraining by the lower inflammation reactivity of depleted hepatic and muscular proteins. FUTURE PROSPECTS AND PROJECTS: Early diagnosis of overtraining diagnosis may be established only from a battery of analyses, which should include the whole of the potential parameters. These remain unpredictable and do not allow systematic determination of new cases. Only a longitudinal study of the physiological situation appears to allow the necessary conditions for detecting overtraining in the early stages of its process for each subject.     

Assessment of blood coagulation in severe liver disease using thromboelastography: use of citrate storage versus native blood.

Thromboelastography evaluates the viscoelastic properties of blood coagulation. Using native blood, measurement must start soon after sampling. With normal coagulation, native and citrated blood values correlate well. No data exists from cirrhotic patients. We compared native and citrate thromboelastography parameters in 30 cirrhotic patients (20 Child-Pugh C class, two liver failure). Thromboelastography was performed within 4 min using native blood and after recalcification within 1-2 h of citrate storage. Thromboelastography variables (, alpha, ) were compared using the Mann-Whitney test, correlation investigated with the Pearson method and the degree of agreement with the Bland-Altman method. There was no significant difference between citrated and native blood for all variables. Median values for native and citrated were, respectively, 16.4 (range 2.3-22.5) and 15.1 (range 9.8-29.9); 6.3 (range 3.5-11.3) and 6.2 (range 2.8-10.9); 48.3 (range 30.7-62.9) and 46.2 (range 30.4-60.4); angle alpha 30.8 (range 18.7-46.8) and 33.2 (range 19.9-55.8). Correlation for each variable was significant ( 0.01). There was a good degree of agreement for all but two patients (both bleeding) for all variables. Citrated blood can substitute native blood using thromboelastography in cirrhotic patients, allowing more time between sampling and the thromboelastography measurement.     

Heparin-induced inhibitory effects of a prothrombin complex concentrate on global tests of haemostasis.

Prothrombin complex concentrates (PCC) have been used as bypassing agents for the treatment of haemophilia A patients with inhibitor as well as for replacement therapy in congenital and acquired deficiencies of vitamin-K-dependent clotting factors. The efficacy of PCC is variable, however, especially during long-term and high-dose use, and all currently available products of this nature contain heparin. We have examined the haemostatic properties of PCC using reconstituted whole blood made by mixing coagulation-factor-deficient plasma and washed blood cells. In rotation thromboelastometry (ROTEM), the recommended therapeutic dose of Proplex ST corrected the abnormal patterns. At higher concentrations, however, the ROTEM patterns regressed. In addition, specific assays of coagulation factors appeared unreliable in the presence of 2.5 U/ml Proplex ST; the abnormalities were corrected when protamine sulfate was added. The findings suggest that the presence of heparin in PCC might have a greater effect on global haemostasis. Careful attention to the anticoagulant effect as well as thrombogenicity of PCC is required. Monitoring therapy using such as ROTEM analysis could be highly informative.     

Outcomes of mentored, grant-funded fellowship training in haemostasis /thrombosis: findings from a nested case-control survey study

Successful strategies by which to effectively recruit and retain academic subspecialists in benign haematology have not been established. To evaluate the effectiveness of a grant-funded, mentored fellowship with respect to retention and early career goals in haemostasis/thrombosis, we sought to compare outcomes for graduates of a grant-funded, mentored fellowship training programme in haemostasis/thrombosis [the National Hemophilia Foundation (NHF)-Baxter Clinical Fellowship Award] during conventional haematology/oncology fellowship training (cases), vs. their training peers who were graduates of conventional haematology/oncology fellowship training alone (controls), via a nested case-control survey study. Survey response rate was 85% (11/13) for cases and 90% (9/10) for controls. All respondents had pursued careers in academic haematology/oncology. Median (range) percent time spent in benign haematology postfellowship was 98% (70-100%) for cases vs. 0% (0-20%) for controls. Time spent in research was significantly greater among cases than controls (median 80% [range: 42-90%] vs. 55% [10-80%], respectively; P = 0.01). By years 3-4 postfellowship, median annual number of peer-reviewed publications was higher for cases than controls (3.5 vs. 1.0; P = 0.01). Cases were also more successful in grant funding (including K-awards). These data suggest that a grant-funded, mentored fellowship training programme in haemostasis/thrombosis may be superior to conventional haematology/oncology fellowship training alone with respect to outcomes of retention in clinical care/research, early-career grant funding and publication productivity.     

Systemic haemostasis after intermittent pneumatic compression. Clues for the investigation of DVT prophylaxis and travellers thrombosis

Intermittent pneumatic compression (IPC) is known to provide effective prophylaxis against post-surgical deep-vein thrombosis (DVT), and other procedures based on reducing venous stasis have been promoted recently to minimize the risk of thromboembolism after long-haul travel ('travellers thrombosis'). This study sought to measure the effects of IPC on systemic haemostasis, which are currently disputed. IPC was applied for 120 min on 21 male, non-smoking volunteers ranging in age from 19 to 47 years. IPC promoted a significant increase in global fibrinolytic potential. Levels of urokinase plasminogen activator activity (uPA) measured using an amidolytic assay were raised after IPC. However, enzyme-linked immunosorbent assays (ELISA) of uPA antigen, and the activities of tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) were not statistically different from those in control experiments. IPC led to highly significant falls in factor VIIa, associated with increased levels of tissue factor pathway inhibitor (TFPI). IPC enhances fibrinolysis and suppresses procoagulant activation. Measurements of specific fibrinolytic components do not reflect overall fibrinolytic activity and are highly dependent on the method of assay. The results provide important clues for detailed studies of the effects of haemodynamics on systemic haemostasis.     

The correlation of in-vitro susceptibility tests with in-vivo results of antibiotic treatment.

The reason why the results of the clinical response to therapy do not always correspond to antibiotic laboratory testing include errors in performance or interpretation of test results, imprecise definition of resistance and host factors which influence the outcome of treatment regardless of the antibiotic being used. These factors are reviewed. Cephalosporins and penicillins pose problems because the prime effects of enzymes in influencing the outcome of treatment are not always clear in standard tests for susceptibility. Inter-laboratory differences in interpretation of tests makes comparisons of tests between centres difficult and the use of reference strains for validation of testing is proposed.     

The Platelet Function Analyzer (PFA-100®): a novel in-vitro system for evaluation of primary haemostasis in children

The PFA-100^® system provides an in-vitro method of assessing primary platelet-related haemostasis by measuring the time (the closure time, or CT) taken for a platelet plug to occlude a microscopic aperture cut into a membrane coated with collagen and either epinephrine or ADP. We used the system to establish normal ranges for CTs in healthy children, adults and neonates. Mean CTs of healthy children were independent of the needle gauge used (21G or 23G) for blood sampling; they were very similar to the mean CTs of healthy adults, but longer than mean CTs of healthy neonates. Although children with haemophilia had normal CTs, the PFA-100 system was found to be potentially useful in screening for von Willebrand disease in children.     

Vascular prostacyclin and plasminogen activator activity in experimental and clinical conditions of disturbed haemostasis or thrombosis.

In several experimental and clinical conditions associated with thrombotic tendency, or complicated by thrombotic episodes, either prostacyclin (which inhibits platelet aggregation) or plasminogen activator (which promotes fibrinolysis), or both, appear to be decreased in the vessel wall. In other conditions, however, either activity may not change or even be increased. Possibly, vascular damage is followed by an early stimulation of both activities (a defence mechanism?) which may be subsequently reduced or exhausted. While the role of vascular plasminogen activator in haemorrhagic conditions is apparently unknown, prostacyclin activity appears to be markedly enhanced both in experimental animals and in patients with uraemia and bleeding complications. There is a suggestive evidence that uraemic plasma powerfully stimulates vascular prostacyclin generation.     

Simultaneous measurement of all thrombosis parameters from native human blood: usefulness in monitoring efficacy and complications of thrombolytic therapy

The sequelae of thrombus formation, both by shear forces and by collagen fiber, the subsequent coagulation, and the dislodgement of thrombi (thrombolysis) were measured from a small volume of nonanticoagulated blood sample, by a new instrument. Addition of streptokinase (SK) or tissue-type plasminogen activator (rt-PA) to the blood sample eliminated the need for anticoagulation for thrombolysis measurement: clot lysis preceded and allowed thrombolysis to occur. The test revealed activation of platelets and coagulation by SK and rt-PA. Apart from this general trend, platelet reactivity in response to plasminogen activator showed great individual variation. Whether the greatly enhanced (42%) or prolonged hemostasis (13%), observed in different blood samples with rt-PA, could be used as a predictor of reocclusion or bleeding complications remains to be established. Thrombolysis did not occur in 12% of the samples tested. These thrombolysis models may be useful for developing new agents for the dissolution of platelet-rich thrombi. It is suggested that this technique be used for monitoring thrombolytic therapy.     

Comparison of various D-dimer tests for the diagnosis of deep venous thrombosis.

Analyses of D-dimers in plasma are frequently used as diagnostic tools for deep venous thrombosis (DVT). Enzyme-linked immunosorbent assays (ELISAs) are considered to be the method of choice for quantitative assays, but are time consuming. Therefore, we have assessed plasma levels of D-dimers in patients with clinically suspected DVT using quantitative (Asserachrom D-Di ELISA and TintElize), semiquantitative (Minutex latex, D-Di latex, NycoCard D-Dimer) and qualitative (INSTANT.I.A) assays. Phlebography was used as the gold standard to verify or exclude the suspected diagnosis. We conclude that the fast assays, INSTANT.I.A and Minutex, have essentially the same negative predictive value [91% and 89%, respectively, using a cut-off value < 0.5 mg/l fibrinogen equivalent units (FEU)] for excluding DVT as the Asserachrom D-Di ELISA and TintElize tests (92%). The D-Di Latex assay had a negative predictive value of 82% (cut-off < 0.5 mg/l FEU) and turned out to be less useful in our material. The NycoCard D-dimer assay had a negative predictive value of 100% when using the cut-off value < 0.5 mg/l FEU, but this was substantially lower when the cut-off was changed to < or = 0.5 mg/l. Thus, we conclude that several fast tests offer a simpler and more rapid way of determining plasma levels of D-dimer than conventional ELISA methods without loss of clinical usefulness in excluding DVT.