Global Hemostasis Tests Research Articles

Изменения сосудисто-тромбоцитарного и коагуляционного гемостаза как предиктор неблагоприятного исхода у новорожденных детей, перенесших асфиксию при рождении

The hemostatic system is complex and evolves continuously since gestation and well into the adult years, in a process known as “developmental hemostasis”. This article presents information about the functioning of the hemostatic system in normal and pathological conditions (birth asphyxia) in newborns, reflects fundamental differences in hemostatic functioning during the neonatal period and the possibilities in maintaining normal hemostasis in conditions of physiological deficiency of both clotting factors and the anticoagulant system. The article highlights various methods of diagnosing the hemostatic system used in neonatology. The so-called global hemostasis tests are being introduced into neonatal practice. The study of hemostasis using thromboelastography technique allows to correct for the patient's real body temperature and estimate both the interaction of platelets and clotting factors and examine the plasma hemostasis component in isolation. The effects of neonatal asphyxia and therapeutic hypothermia procedures on the hemostatic system are poorly understood. This review article attempts to summarize the data available in the world scientific literature concerning this problem. Key words: newborns, developing hemostasis, asphyxia, hypoxic-ischemic encephalopathy, thromboelastography, coagulation, therapeutic hypothermia

Predicting bleeding after liver biopsy using comprehensive clinical and laboratory investigations: A prospective analysis of 302 procedures

BackgroundLiver biopsy carries a small risk of bleeding complications. No validated clinical or laboratory tool helps predict liver biopsy–related bleeding. ObjectivesTo determine whether global hemostasis tests and/or a clinical questionnaire could identify patients at risk of liver biopsy–related bleeding. Patients/MethodsConsecutive patients scheduled for liver biopsy with an overnight hospital stay were prospectively included. Before liver biopsy, routine hemostasis tests, Platelet Function Analyzer 100, thromboelastometry, thrombin generation assay, plasma clot lysis time, and a clinical questionnaire were performed. Bleeding was defined as a liver hematoma or new free fluid on a systematic ultrasound performed 24 h after liver biopsy or a decrease in hemoglobin level of 2 g/dL or more in patients with pre‐existing free fluid in the abdominal cavity. ResultsThree hundred two patients were included: 173 underwent percutaneous and 129 transjugular liver biopsy. There were 21 bleeding episodes (7%); 20 based on ultrasonographic criteria, 1 on laboratory criteria. None of the hemostasis tests and no item of the clinical questionnaire were associated with liver biopsy–related bleeding in the overall study group. Same results were obtained in subgroup analyses focusing on patients who underwent percutaneous liver biopsy, transjugular liver biopsy, or on patients with cirrhosis. Pain 2 h after liver biopsy was more frequent in patients with liver biopsy–related bleeding (55% vs. 23% p = .002). ConclusionsAn extensive hemostasis workup, including global hemostasis assays, does not improve prediction of liver biopsy–related bleeding. Pain 2 h after liver biopsy should alert the clinician to the possibility of procedure‐related bleeding.

In seeking diagnostic tool for laboratory monitoring of FXII-targeting agents, could assessment of rotational thromboelastometry (ROTEM) in patients with factor XII deficiency be useful?

Introduction: Targeting factor XII (FXII) is a new concept for safe thrombosis prophylaxis. Global hemostasis tests offer promise in terms of the laboratory monitoring of FXII inhibition. The present study examines selected parameters of rotational thromboelastometry (ROTEM) in patients with FXII deficiency. The objective of this study was to assess the impact of FXII deficiency on selected parameters of ROTEM, which can be significant in the laboratory monitoring of FXII inhibition. Material and methods : The study included 20 patients with FXII deficiency ≤40% and 21 volunteers free of it. Clotting time (CT), clot formation time (CFT), alpha angle (α), maximum clot firmness (MCF), and maximum lysis (ML) were recorded in ROTEM. Results: For the INTEM test, CT and CFT readings were markedly higher in FXII deficient patients than in controls. No marked differences in relation to MCF and ML were found. Conclusion: The results of ROTEM show that FXII deficiency has a great impact on the initiation and amplification of coagulation. This was confirmed by a number of marked correlations between FXII activity and certain ROTEM parameters. ROTEM tests merit further investigation as treatment control strategies in the context of FXII inhibition.

Clinical utility of activated partial thromboplastin time clot waveform analysis and thrombin generation test in the evaluation of bleeding phenotype in Hemophilia A.

Hemophilia A is classified as mild, moderate, and severe based on Factor VIII levels (FVIII). Clot-based assays only detect initiation of thrombin generation, hence FVIII levels may not accurately predict the bleeding risk in all hemophilia patients. The entire process of thrombin generation as measured by global hemostasis tests like activated partial thromboplastin time clot waveform analysis (APTT CWA) and thrombin generation test (TGT) may reflect the actual bleeding phenotype. To assess the utility of TGT and CWA as a screening tool to identify bleeders and to evaluate the bleeding phenotype in Hemophilia A. Prospective, observational study of 147 consecutive patients referred for coagulation workup. Bleeding assessment tool was used to identify bleeders. Patients were classified as severe and nonsevere bleeders based on clinical criteria. TGT was performed by calibrated automated thrombogram, CWA by photo-optical coagulometer and factor levels by one stage clot-based assays. The Kruskal-Wallis test with post-hoc analysis was done to examine the difference in CWA/TGT parameters amongst hemophilia classified by FVIII levels. Receiver operating characteristic (ROC) analysis was performed to estimate the diagnostic accuracy of CWA and TGT in discriminating between clinically severe vs nonsevere bleeders. Using ROC derived cut-offs of min1, min2 and peak height of thrombin (PH), the sensitivity (min1:91.67%, min2:91.67%, PH: 97.22%, FVIII: 86.11%) and specificity (min1:100%, min2:100%, PH: 90.91%, FVIII: 90.91%) of CWA/TGT was superior to FVIII to distinguish between clinically severe vs nonsevere bleeders. Phenotypic heterogeneity of bleeding severity was identified in our study population. Clinical severity correlated with CWA/TGT parameters instead of FVIII levels. CWA and TGT are more effective tools than conventional factor assays to identify clinically severe bleeders and tailor prophylaxis as per bleeding phenotype.

A Novel Nonsense Mutation in FGB (c.1421G>A; p.Trp474Ter) in the Beta Chain of Fibrinogen Causing Hypofibrinogenemia with Bleeding Phenotype.

Congenital hypofibrinogenemia is a rare bleeding disorder characterized by a proportional decrease of functional and antigenic fibrinogen levels. Hypofibrinogenemia can be considered the phenotypic expression of heterozygous loss of function mutations occurring within one of the three fibrinogen genes (FGA, FGB, and FGG). Clinical manifestations are highly variable; most patients are usually asymptomatic, but may appear with mild to severe bleeding or thrombotic complications. We have sequenced all exons of the FGA, FGB, and FGG genes using the DNA isolated from the peripheral blood in two unrelated probands with mild hypofibrinogenemia. Coagulation screening, global hemostasis, and functional analysis tests were performed. Molecular modeling was used to predict the defect of synthesis and structural changes of the identified mutation. DNA sequencing revealed a novel heterozygous variant c.1421G>A in exon 8 of the FGB gene encoding a Bβ chain (p.Trp474Ter) in both patients. Clinical data from patients showed bleeding episodes. Protein modelling confirmed changes in the secondary structure of the molecule, with the loss of three β sheet arrangements. As expected by the low fibrinogen levels, turbidity analyses showed a reduced fibrin polymerisation and imaging difference in thickness fibrin fibers. We have to emphasize that our patients have a quantitative fibrinogen disorder; therefore, the reduced function is due to the reduced concentration of fibrinogen, since the Bβ chains carrying the mutation predicted to be retained inside the cell. The study of fibrinogen molecules using protein modelling may help us to understand causality and effect of novel genetic mutations.

Выбор теста тромбоэластометрии для оценки эффекта профилактической дозы низкомолекулярных гепаринов

Цель исследования: изучить возможность использования тестов тромбоэластометрии (ТЭМ) для оценки эффекта профилактической дозы низкомолекулярных гепаринов (НМГ). Материалы и методы. В исследование включено 30 пациентов (14 мужчин и 16 женщин), которым выполнялась резекция печени в связи с метастатическим поражением колоректальным раком. Для профилактики тромботических осложнений в послеоперационном периоде использовали НМГ надропарин. У пациентов в первую неделю послеоперационного периода определяли активность антитромбина III (AT-III) и проводили ротационную ТЭМ, при которой исследовали 3 пробы крови: проба № 1 — утром натощак перед введением пациенту НМГ; проба № 2 — как и первая проба, но с добавлением в пробу НМГ in vitro до достижения целевой концентрации 0,5 МЕ / мл крови; проба № 3 — через 4 ч после подкожного введения НМГ. У 10 пациентов проведены тесты ТЭМ — INTEM и NATEM (нативный тест), в которых оценивали показатели CT, CFT, угол α, MCF; у 20 пациентов проведен только тест NATEM. Результаты. Средние показатели теста INTEM перед введением НМГ не выходили за рамки референтных значений, в тесте NATEM только показатель MCF превышал нормальные значения. После внесения в исследуемую пробу 0,5 МЕ / мл НМГ in vitro средние значения CT и CFT теста ТЭМ значимо удлинялись. Интервал CT теста INTEM был удлинен выше нормы в 2 наблюдениях, CFT теста INTEM превышал норму в 1 наблюдении. В тесте NATEM удлинение интервалов CT и CFT выше нормальных выявлено в 100% случаев. Показатели ТЭМ через 4 ч после введения НМГ были аналогичны результатам in vitro. Выявлены статистически значимые различия показателей нативного теста от активности AT-III, наибольшей чувствительностью и специфичностью из них обладали интервал CT и ΔCT. Заключение. Метод ротационной ТЭМ является перспективным прикроватным методом оценки антикоагулянтного эффекта профилактических доз НМГ. Использование этого метода в послеоперационном периоде позволит индивидуально дозировать антикоагулянты и повысить эффективность профилактики венозных тромбоэмболических осложнений. Objectives: to investigate the applicability of various types of thromboelastometry (TEM) to assess effects of low molecular weight heparins (LMWHs) administered in prevention doses. Patients/ Methods. The study included 30 patients (14 men and 16 women) who underwent liver resection due to metastatic lesions of colorectal cancer. LMWH (nadroparin) was used to prevent thrombotic complications in the postoperative period. In the first week of postoperative period antithrombin III (AT-III) activity was determined and rotational TEM was performed, in which 3 blood samples were examined: sample № 1 — in the morning, fasted, before LMWH administration; sample № 2 — like the first sample, but with in vitro LMWH addition to the sample to achieve the target concentration of 0.5 IU per 1 ml of blood; sample № 3–4 hours after subcutaneous LMWH administration. TEM tests — INTEM and NATEM (native test) were performed in 10 patients with CT, CFT, angle α, and MCF assessment; in 20 patients only NATEM test was performed. Results. The average values of INTEM test before LMWH administration were within the reference values, in NATEM test only MCF exceeded normal values. The average CT and CFT values in TEM test significantly increased after in vitro LMWH addition to the test sample. Elongation of CT interval in INTEM test was found in 2 cases, CFT in INTEM test exceeded the norm in 1 case. Elongation above normal of CT and CFT intervals in NATEM test was noticed in 100% of cases. TEM parameters in 4 hours after LMWH administration were similar to in vitro results. The statistically significant differences between native test parameters and AT-III activity were revealed; CT and ΔCT intervals had the greatest sensitivity and specificity. Conclusions. Rotational TEM is a perspective bedside method for anticoagulant effect assessment of LMWH prevention doses. Using this method in postoperative period will allow to dose anticoagulants individually and increase prophylaxis effectiveness of venous thromboembolic complications.

Primary Hemostasis in Chronic Liver Disease and Cirrhosis: What Did We Learn over the Past Decade?

Changes in primary hemostasis have been described in patients with chronic liver disease (CLD) and cirrhosis and are still subject to ongoing debate. Thrombocytopenia is common and multifactorial. Numerous studies also reported platelet dysfunction. In spite of these changes, primary hemostasis seems to be balanced. Patients with CLD and cirrhosis can suffer from both hemorrhagic and thrombotic complications. Variceal bleeding is the major hemorrhagic complication and is mainly determined by high portal pressure. Non portal hypertension-related bleeding due to hemostatic failure is uncommon. Thrombocytopenia can complicate management of invasive procedures in CLD patients. Recently, oral thrombopoietin agonists have been approved to raise platelets before invasive procedures. In this review we aim to bundle literature, published over the past decade, discussing primary hemostasis in CLD and cirrhosis including (1) platelet count and the role of thrombopoietin (TPO) agonists, (2) platelet function tests and markers of platelet activation, (3) von Willebrand factor and (4) global hemostasis tests.

Hyper-coagulability in obese patients accurately identified by combinations of global coagulation assay parameters

BackgroundRoutine laboratory methods are insensitive to hyper-coagulation, which may be detected by global hemostasis tests. Calibrated Automated Thrombogram (CAT) is a gold standard method to measure thrombin generation and coagulation potential. Thrombodynamics (TD) is a new global assay that monitors the spatio-temporal propagation of blood coagulation, separating initiation from amplification/propagation phases of coagulation and visualizing fibrin clot formation. AimWe investigated whether CAT and/or TD can identify hyper- and hypo-coagulable states in patients with well-characterized phenotypes and which parameters could be used as potential predictors of thrombotic risk. MethodsBlood was collected from: (1) forty healthy volunteers; (2) twelve obese patients scheduled for bariatric surgery (BMI ≥ 35 kg/m2); (3) nine patients under therapy with vitamin K-antagonists (median INR 2.7); (4) eight patients treated with low molecular weight heparins (anti-Xa activity between 0.5 and 0.7 U anti-Xa/ml); (5) ten patients with hemophilia A or B. Tissue factor induced thrombin generation was measured with CAT. Propagation of thrombin generation and clot growth from a tissue factor coated surface were monitored with TD. ResultsThrombin generation and fibrin clot formation parameters were significantly higher in obese patients compared to healthy volunteers and anticoagulated or hemophilic patients. ROC analysis of combined CAT or CAT/TD parameters (integrating thrombin generation and fibrin clot formation) demonstrated an excellent accuracy in detecting hyper-coagulability. ConclusionCombinations of CAT assay parameters and of parameters of thrombin generation burst with final fibrin clot properties allow recognizing accurately hyper-coagulable plasma and may represent predictive markers for thrombotic events.

Predictability of the Caprini Risk Assessment Model for Postoperative Venous Thromboembolism may be Increased by Global Test of Haemostasis

Introduction - Postoperative venous thromboembolism (VTE) is still a challenge. Standard preventive protocol, that includes elastic compression stockings and low-molecular-weight heparin (LMWH) injection, may be not equally effective in different patients at high risk. Previously it has been shown that patients with 11 and more Caprini scores have a dramatically increased risk of VTE at the background of elastic compression and LMWH injections. The way to improve such situation is to understand, why standard prophylaxis does not work properly in some individuals. To achieve this goal may be used modern global tests of hemostasis. The aim of this study was to compare predictability for postoperative deep vein thrombosis (DVT) of the classic Caprini 2005 RAM and its modified version, considering the results of TD test in surgical patients at high risk. Methods - This was a prospective observational clinical study involving 80 patients (33 men and 47 women, mean age - 73,9±7,2 years), undergoing major (79 cases) or minor (1 case) surgery for colorectal cancer. Patients were at high risk of postoperative venous thromboembolism: 5-15 Caprini scores (mean - 9,9±2,0) and received combined prophylaxis: anti-embolic above-knee compression stockings and LMWH injections (enoxaparin 40 mg once daily) until discharge. LMWH was administered in a fixed hour according to the time of blood shambling for TD. Before and after the surgery (0 day, 5-7 day and 1 month) duplex ultrasound was performed to detect postoperative DVT. Results - DVT was found in 21 of 80 patients (26.3%; 95% CI: 17,9-36,8%) at 5-7 day after surgery. No new venous thromboembolism events were detected at 1 month. Regression analysis and ROC-curve showed a significant predictability of Caprini scores for DVT (p<0,0001, S=0,839±0,045). Analysis of ROC-curve coordinates showed a cut-off point of 11 scores, which with the sensitivity of 76.2% and a specificity of 74.6% predicted DVT. The results of TD test showed significant hypercoagulation at the background of LMWH administration in patients with DVT. Regression analysis and ROC-curves demonstrated that TD parameters as stationary velocity of clot growth, measured at 2 hours after surgery (Vst), and initial velocity of clot growth, measured 24 hours after the LMWH injection (Vin), had the greatest predictability for postoperative DVT: S=0,764 ± 0,109, p=0,049 and S=0,946±0,047, p=0,001, respectively. The cut-off points for DVT prediction with high sensitivity/specificity appeared to be Vst>32,5 (normal range 20-29) μm/min and Vin>64,5 (normal range 36-56) μm/min. Identified thresholds for TD parameters have been integrated into Caprini RAM under the item "other congenital or acquired thrombophilia" (score of 3). Total Caprini scores were recalculated in patients who had one or both TD parameters exceeded the cut-off, followed by re-analysis of the ROC-curves. It was found that the Caprini RAM with integrated Vin>64,5 μm/min characterized by the highest predictability for DVT (S=0,876±0,038, p<0,0001) and increased cut-off up to 12 scores with sensitivity of 85,7% and specificity of 72.9%. Conclusion - The integration of TD parameters into Caprini RAM allows increasing predictability for postoperative DVT.

The hemostasis system in children with hereditary spherocytosis

IntroductionPatients with hereditary spherocytosis (HS) are characterized by having an increased risk for thrombosis. An early manifestation of thrombotic complications can occur even in childhood, especially after surgery. Hypercoagulability can be associated with hemolytic crises. AimThe aim of this study was to investigate the hemostatic state in children with HS using global hemostasis assays. MethodsThe hemostatic status of 62 children (38 boys and 24 girls; age range: 0.5 to 17 years) with HS during and without hemolytic crisis was assessed using clotting times (APTT, TT, and PR), fibrinogen and D-dimer levels, and global hemostasis, thromboelastography (TEG) and thrombodynamics (TD) assays. One hundred and two healthy children undergoing annual medical examination were enrolled as a control group. ResultsTEG and TD parameters were increased in the children with HS compared to the control group (60 ± 5 mm vs. 53 ± 4 mm, p < 0.05 for TEG maximum amplitude; 28 ± 3 μm/min vs. 24 ± 2 μm/min, p < 0.05 for TD clot growth rate), while APTT, TT and PR were not significantly different between the two groups. Patients with HS were divided into 2 groups: those during hemolytic crisis (28 patients) and those without hemolytic crisis (34 patients). TEG and TD parameters were increased in those during hemolytic crisis compared to the steady state HS group (62 ± 5 mm vs. 57 ± 4 mm, p < 0.05 for TEG maximum amplitude; 31 ± 4 μm/min vs. 26 ± 3 μm/min, p < 0.05 for TD clot growth rate). The D-dimer levels were increased in 4 HS patients, for whom the activation of blood clotting was noted. Fibrinogen levels were decreased in patients with HS compared to the control group (2.1 ± 0.4 mg/ml vs. 2.6 ± 0.4 mg/ml, p < 0.05). Other tests were within the reference ranges for both groups. ConclusionsThe global hemostasis tests TEG and TD revealed hypercoagulability in patients with HS. More dramatic changes were observed in patients experiencing a hemolytic crisis.

Platelet Dysfunction and Cellular Microparticles May be Involved in the Hipercoagulable State Observed in Obstructive Sleep Apnea Syndrome

Introduction: Obstructive sleep apnea syndrome (OSA) is a common disorder characterized by repetitive episodes of nocturnal breathing cessation due to upper airway collapse that cause intermittent hypoxia. Because of this, there is an increase in oxidative stress, endothelial damage and mortality associated with the incidence of thrombotic events. The evaluation of platelet function by flow cytometry (FCM) and hemostatic capacity through global hemostasis tests have been successfully used in the study of the prothrombotic state associated to numerous diseases. We anticipate that these techniques may help to clarify the mechanism involved in the prothrombotic state of OSA.Materials and Methods: We included 16 OSA patients diagnosed in the Pneumology Unit of the University Hospital La Paz and 59 healthy controls recruited in the Donor Unit of the same Hospital.Platelet surface receptors were analysed by FCM (FACScan, BD Biosciences) with specific monoclonal antibodies (mAbs) against CD41/αIIb and CD61/β3, CD42a and CD42b. Platelet activation was assessed by FCM through PAC1-FITC binding (mAbs that recognizes activated conformation of fibrinogen receptor), P-selectin and CD63 exposure in baseline condition and after stimulation with 100 µM TRAP (agonist of the PAR-1 receptor) and 10 µM ADP. Platelet apoptosis was analysed by FCM through FITC-annexin V (BD Pharmingen) binding to phosphatidylserine (PS) exposed on platelet membrane under basal conditions and by determination of activity of caspases -3/7, -8, and -9 (BD Millipore).The overall hemostatic capacity was determined by the evaluation of thrombin generation using the Calibrated Automatic Thrombinography (CAT) with fresh platelet-poor plasma (obtained after double centrifugation of whole blood at 2500g, 20 minutes and 24ºC) and by the assessment of kinetics of clot formation using Rotational Thromboelastometry (ROTEM® Gamma, Tem Innovations GmbH, Spain) using whole blood and activation by recalcification (naTEM® test, Tem Innovations GmbH, Spain).The statistical analysis was performed using SPSS 9.0 software (SPSS Inc., Chicago, Illinois, USA). Statistical significance was established at p <0.05.Results: No significant differences in age or sex were found between groups. Patients presented anxiety-depressive disorder (15%), ischemic cardiomyopathy (8%), dyslipidemias (38%), arterial hypertension (69%) and lower minimum oxygen pressures during sleep (mean±SD; patients: 76±8 % vs controls: 89±1 %; p< 0.001) indicating hypoxia. In basal conditions, an increase of CD42a subunit of the von Willebrand factor receptor was observed in patients with OSA but no differences were found in the exposure of fibrinogen receptor either at basal condition or after activation with TRAP or ADP (Table 1 and Figure 1). Platelet PS exposure before and after TRAP stimulation was higher in the patient group (Figure 1) but no differences between groups were observed in caspase activities (Table 2). Clotting time (CT), maximum clot firmness (MCF) and MP-associated thrombin generation were higher in patients with OSA (Figure 2).Conclusion: Mechanisms related to the mobilization of P-selectin to platelet surface and with the maintenance of phospholipids distribution in platelet membrane might be altered in OSA. The increment of P-selectin exposure may favor the interaction between white cells and platelets through PSGL-1 ligand from lymphocytes which in turn may produce endothelial damage explaining part of OSA's pathophysiology. A lower CT and higher MCF reflected a hypercoagulable state in patients, perhaps related to their higher MP-associated thrombin generation and, at least in part, due to the enhanced platelet-PS exposure. Our results also suggest that both ROTEM and CAT are useful tools for characterizing prothrombotic states in OSA patients.Work supported by grant from FIS-FEDER PI15/01457. NB holds a Miguel Servet II (FIS-FEDER CP14/00024). DisclosuresÁlvarez-Roman:Shire: Consultancy; NovoNordisk: Consultancy; SOBI: Consultancy. Jimenez-Yuste:Grifols: Consultancy, Research Funding; Octapharma: Consultancy, Research Funding; CSL Behring: Consultancy; Bayer: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Shire: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; NovoNordisk: Consultancy, Research Funding; Sobi: Consultancy, Research Funding. Butta:FIS-Fondos FEDER: Research Funding.

Biological variation estimates of prothrombin time, activated partial thromboplastin time, and fibrinogen in 28 healthy individuals.

Although tests of global hemostasis prothrombin time (PT) and activated partial thromboplastin time (aPTT) should not be used for prediction of bleeding risk, these tests are often used by many clinicians in daily practice particularly as a preoperative screening test. Robust biological variation (BV) data are needed for safe clinical applications of these tests. In this study, a stringent protocol was followed to estimate the BV's for PT, aPTT, and fibrinogen levels. Weekly blood samples were obtained from 28 healthy individuals (18 females, 10 males) during 10weeks study period. All measurements were performed with Stago STA-R coagulation analyzer. Prior to coefficient of variation (CV)-analysis of variance (ANOVA), the data were assessed for normality, trends, outliers, and variance homogeneity. Sex-stratified within-individual (CVI ) and between-individual (CVG ) BV estimates were determined for PT, aPTT, and fibrinogen tests. No difference was found between male and female estimates of BV. The observed CVI and CVG estimates were found to be lower than those previously published. Only for fibrinogen, CVI was higher than CVG . Following a meticulous protocol, our study results provide up-to-date and more stringent BV estimates of global hemostasis tests.

Mechanisms and management of coagulopathy in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is a subtype of leukemia which is associated with unique and distinctive coagulopathy. In the absence of treatment it is rapidly fatal and even after initiation of therapy the major cause of early mortality is related to hemorrhagic complications. The coagulopathy can be exacerbated with the start of treatment. In the absence of early hemorrhage related deaths the probability of cure exceeds 90% in low and intermediate risk patients and 80% even in high risk patients, highlighting the importance of understanding the pathophysiology of this complication and instituting prompt and appropriate management strategies. The coagulopathy in APL is complex and results from a combination of thrombocytopenia, disseminated intravascular coagulation and hyperfibronlysis. Recently the effect of all-trans retinioc acid (ATRA) induced ETosis on exacerbating coagulopathy in the first few days after starting therapy with this agent raises the potential for potentially novel strategies to reduce the risk of hemorrhage. Currently management is mainly related to rapid initiation of therapy with ATRA along with appropriate and adequate replacement of blood products to correct the coagulopathy. There is limited role for the use of low dose anti-coagulants and anti-fibrinolytic agents in the initial management of this disease. There is limited data on the use of rFVIIa or the use of global tests of hemostasis in the management of this condition.

Cupincin: A Novel Cupin Domain Containing Protease from Rice (Oryza sativa L.) Bran Comprising of Procoagulant and Fibrinogenolytic Activity.

The current study was carried out to evaluate the pharmacological properties of cupincin- A novel cupin domain containing metalloprotease with limited proteolysis from rice bran on blood coagulation and hydrolysis of human fibrinogen. Cupincin preferentially hydrolyzed the Aα chain of fibrinogen and then the Bβ-chain, but not the γ-chain. Cupincin reduced the re-calcification time of citrated human plasma dose dependently. Analysis of citrated whole blood in the presence of cupincin by rotem showed a decrease in coagulation time and clot formation time. Sonoclot analysis indicated that cupincin cleaved fibrinogen of whole citrated blood. SDS-PAGE and sonoclot analysis (LI-30) indicated that cupincin lacked plasmin-like activity. Global hemostasis tests like rotem and sonoclot analysis determined cupincin as a procoagulant enzyme. Cupincin did not show any effect on prothrombin time and activated partial thromboplastin time tests suggesting its action on the common pathway of coagulation. The involvement of proteases from rice (Oryza sativa L.) in haemostasis has never been exploited before. This study could provide the basis for the development of new procoagulant agents from a nontoxic source like rice.

Hemostatic Complications in Hepatobiliary Surgery.

AbstractHepatobiliary surgery is a well-known risk factor for thrombotic complications but is also associated with substantial perioperative blood loss. Given the central role of the liver in hemostasis, hepatobiliary surgery is frequently accompanied by complex changes in the hemostatic system. Increasing knowledge of these changes has resulted in an improved understanding of the etiology of some of the hemostatic complications. In the early postoperative period a prolongation of conventional coagulation test times, such as the prothrombin time, is frequently seen. Together with a decreased platelet count, this suggests a hypocoagulable state. The concomitant decline of anticoagulant factors and development of a von Willebrand factor/ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) imbalance, however, suggest a hypercoagulable state, potentially contributing to the risk of thromboembolism. Postoperative thromboprophylaxis should be initiated early to avoid thrombosis, and intensified prophylaxis might benefit high-risk patients. The risk of hemorrhagic complications during hepatobiliary surgery has diminished over time, mainly due to improved surgical and anesthesiological techniques. However, bleeding can still be profound in individual patients and is difficult to predict using (global) hemostasis tests. A restrictive transfusion and fluid infusion policy to maintain a low central venous pressure is crucial in prevention of perioperative bleeding. However, when active bleeding occurs, proactive prohemostatic management is required.

The Management of Factor XI Deficiency in Pregnancy.

Management of factor XI (FXI) deficiency in pregnancy is complicated by lack of correlation between FXI level and bleeding risk. Clinicians should be vigilant about the potential for prolonged or excessive bleeding following miscarriage or termination of pregnancy, or postpartum hemorrhage (PPH). A multidisciplinary approach along with an individual care plan is recommended to prevent bleeding complications. Assessment of bleeding history, FXI level, and global tests of hemostasis can aid management decisions regarding hemostatic prophylaxis. The risk of PPH can be minimized by obstetric measures to avoid uterine atony and genital trauma, in addition to provision of appropriate hemostatic prophylaxis for labor and delivery. Women with FXI deficiency can be given the option of regional anesthesia, provided that prior consideration has been given to assessment of potential bleeding risk and appropriate treatment strategies are implemented. Antifibrinolytic agents are effective for the majority of women with FXI deficiency, but those with severe deficiency/phenotype require FXI concentrate. Recombinant activated factor VII (rFVIIa) has also been used successfully to prevent bleeding in FXI deficiency. However, all treatments should be used with caution in pregnancy due to thrombogenic potential. Neonatal bleeding complications are rare in FXI deficiency; however, hemostatic assessment and cover are important for invasive procedures such as circumcision.

PT, aPTT, TT and the hemostatic safety threshold of dabigatran and rivaroxaban.

The proposals of the Working group on perioperative hemostasis (Groupe d'intérêt en hémostase péri-opératoire (GIHP)) concerning the perioperative management of patients receiving the direct oral anticoagulants (DOACs) are based on the measure of their anticoagulant activities (anti-Xa for rivaroxaban and anti-IIa for dabigatran) with a safety threshold ≤ 30 ng/mL. If the dosage of the drug is not available, proposals are based on the combination of a PT ≥80% and an aPTT ≤1.20. The aim of our study was to evaluate the performance of PT, aPTT and thrombin time to predict values above or below the safety threshold. The measurement of DOACs concentration was carried out in 64 samples from patients treated with rivaroxaban and 48 samples from patients treated with dabigatran. The PT and aPTT were measured for all samples, while the TT was measured only for patients receiving dabigatran. The absence of agreement between the global hemostasis tests and the DOACs concentrations was observed for 10% of patients receiving dabigatran and 27% of patients with rivaroxaban treatment. Apart from dabigatran for which the predictive negative value of PT and aPTT or TT allows to exclude a concentration >30 ng/mL in 100% of cases, our results highlight the risk of misinterpretation when using global coagulation tests (PT and aPPT) for determination of the safety threshold for patients receiving the DOACs.

Thromboelastography, thrombin generation test and thrombodynamics reveal hypercoagulability in patients with multiple myeloma

Patients with multiple myeloma (MM) are at increased risk of venous thromboembolism. Therefore, adequate laboratory control of hemostasis and subsequent adjustments of anticoagulant therapy are necessary. We studied hemostasis changes using thromboelastography (TEG), thrombin generation test (TGT) and thrombodynamics (TD) in primary MM patients (PMMpt, n = 25) and patients in remission (RMMpt, n = 34) during blood stem cell (BSC) mobilization. TD and TEG reveal hypercoagulability in PMMpt (*p < 0.05) in relation to healthy volunteers. There was no difference in any of the tests between PMMpt and RMMpt. We detected no heparin effect in 22% of patients one day after the onset of the prophylactic heparin treatment (500 IU/h) during BSC mobilization; tests shifted toward the hypercoagulability in 75% of patients one day after cyclophosphamide (4 g/m2) chemotherapy. Global hemostasis tests were in good agreement with each other, revealed hypercoagulability and heparin “resistance” in patients with MM and may be useful for therapy individualization.

Preserved clot formation detected by the Thrombodynamics analyzer in patients with cirrhosis

IntroductionPatients with cirrhosis have substantial alterations in their hemostatic system, which are paradoxically associated with the risk of both bleeding and thrombotic complications. However, it still remains difficult to predict those risks, because results from conventional coagulation tests, such as the prothrombin time (PT) and activated partial thromboplastin time (APTT), do not reflect the complex hemostatic changes in these patients. More sophisticated global hemostasis tests, such as thrombin generation assays, are not standardized for routine use yet. Here we examined the spatial clot growth in plasma from patients with cirrhosis using the novel Thrombodynamics assay, which uses a fundamentally new approach to test plasma hemostatic capacity. Materials and MethodsThrombodynamics assays were performed in plasma from thirty-one patients with cirrhosis and twenty-five healthy controls. Results were compared to results with thrombin generation testing and PT/APTT test results. ResultsRates of clot growth, clot size, and clot density from the Thrombodynamics assay were comparable between patients and controls. Thrombin generation in the presence of thrombomodulin was increased in the patients, despite prolonged PT and APTT test results. There was little correlation between parameters derived from the Thrombodynamics assay and the PT, APTT, or thrombin generation data. ConclusionsThe Thrombodynamics assay showed preserved clot formation in plasma from patients with cirrhosis, which is in line with the results of the thrombin generation assay in this study and previously reported by others.

A general review of major global coagulation assays: thrombelastography, thrombin generation test and clot waveform analysis.

Thrombosis and hemorrhage are major contributors to morbidity and mortality. The traditional laboratory tests do not supply enough information to diagnose and treat patients timely and according to their phenotype. Global hemostasis tests might improve this circumstance. The viscoelastic tests (ROTEM/TEG) demonstrated to ameliorate treatment of acute hemorrhage in terms of decreased amount of transfusion and lowered costs. Thrombin generation measurement is indicative for thrombosis and might also become an important tool in managing hemorrhage. While the clot waveform analysis is less well known it could be of worth in staging sepsis patients, early detection of DIC and also in diagnosis and treatment monitoring of hemophiliac patients. Although in different degree all three methods still need more background, standardization and acceptance before a wide clinical application.