The sthemO 301 is a new hemostasis analyzer developed by Diagnostica Stago, combining simultaneously three different methodologies (i.e. clotting, chromogenic and immunological assays) on a single platform. The objective of this evaluation was to assess the analytical performances of the sthemO 301 and to compare it with the STA R Max on a selection of 20 hemostasis parameters (PT, aPTT with two reagents, Clauss fibrinogen, thrombin time, D-dimer, anti-Xa, factors II, V, VII, X, VIII, IX and XI, antithrombin, protein C and protein S). Within-run and inter-laboratory precisions have been evaluated using quality controls. Method comparisons were performed using a minimum of 100 plasma samples, collected across multiple sites, and compared to the STA R Max analyzer. Reference intervals were determined or verified based on published data. Within-run and inter-laboratory precisions were respectively below 5.2 and 7.6% whatever the parameter and the sample used. Pearson correlation coefficient was above 0.976 for all assays and slopes ranged from 0.94 to 1.07 for all but thrombin time, which was at 0.86. Bias at levels of interest were less than 6% for most routine coagulation parameters, coagulation factors and inhibitors. We reported the precision, accuracy and reference intervals of 20 parameters on the sthemO 301, half of these being reported for the first time, which include anti-Xa, factors II, V, VII, X, VIII, IX, XI, protein S free antigen and activity. The sthemO 301 analyzer offers satisfactory analytical performance and a good comparability with the STA R Max for all parameters.
INTRODUCTION:More than 10% of severe hemophilia A patients are paradoxically mild bleeders despite factor VIII (FVIII) levels being <1 IU/dl. Quantitation of FVIII levels <1 IU/dl is a challenge due to the detection limits of traditional one-stage activated partial thromboplastin time, two-stage chromogenic assay, and tissue factor-initiated thrombin generation assay (TF-TGA), impeding a precise characterization of the bleeding phenotype in severe hemophilia patients. OBJECTIVE:The study aimed to enhance the sensitivity of TGA by modifying the trigger reagent for FVIII measurement. METHODOLOGY:We optimized the fluorometric quantitation of thrombin generation triggered by factor IXa (FIXa) in artificial reconstituted plasma by blending varying proportions of normal pooled plasma (NPP) and FVIII-immunodepleted plasma containing a normal level of von Willebrand factor. RESULTS:FIXa-initiated thrombin generation depended on FVIII level, while TF could bypass FVIII deficiency to activate thrombin, making TF-TGA incapable of quantifying low levels of FVIII. When triggering with 0.6 nM FIXa, in the presence of 4 μM phospholipids and 40 μg/ml corn trypsin inhibitor, thrombin generation was highly dependent on FVIII levels even below 1 IU/dl. Among the five routinely used TGA parameters, peak thrombin, endogenous thrombin potential, and velocity index demonstrated the strongest linear correlation with FVIII levels down to 0.1 IU/dl. CONCLUSION:The study evaluated the performance of a modified TGA activated with FIXa. In addition to traditional TF-TGA, FIXa-TGA may provide additional information when assessing the bleeding tendency of patients with severe hemophilia A, facilitating personalized factor replacement therapy.
OBJECTIVE:This study identifies and characterizes the novel compound heterozygous mutations in the KNG1 gene responsible for severe high molecular weight kininogen (HMWK) deficiency in a 3-year-old Chinese boy. METHODS:The proband was identified during preoperative screening due to an isolated, prolonged activated partial thromboplastin time (APTT) without bleeding symptoms. Coagulation profiles, including thromboelastography (TEG), were analyzed. HMWK antigen (HMWK:Ag) levels were quantified by ELISA. Genetic analysis was performed using whole-exome and Sanger sequencing of the KNG1 gene in the proband and his parents. The pathogenicity of the novel variant was assessed according to ACMG/AMP guidelines. RESULTS:Coagulation tests revealed a significantly prolonged APTT and a delayed R time on TEG, which was not corrected with extended incubation. HMWK:Ag levels were severely reduced (1.7 μg/ml) in the proband. Genetic analysis identified compound heterozygous mutations in KNG1: a novel missense variant in exon 5 (c.611C>T, p.Thr204Met) and a known nonsense variant in exon 6 (c.718C>T, p.Arg240*). Bioinformatic tools predicted the p.Thr204Met variant to be deleterious, and it was classified as "Likely Pathogenic." The mutations were inherited in trans, confirming the genetic basis of the HMWK deficiency. CONCLUSION:We report a novel compound heterozygous mutation (c.611C>T and c.718C>T) in the KNG1 gene causing severe HMWK deficiency. This case expands the mutational spectrum of this ultra-rare disorder and highlights that its primary clinical significance is an isolated APTT prolongation without a bleeding diathesis. Genetic diagnosis is crucial to avoid unnecessary treatments, surgical delays, and exposure to blood products in such patients.
Late-onset vitamin K deficiency bleeding (VKDB) usually manifests as intracranial or gastrointestinal hemorrhage, and hematuria is rarely reported. We describe a 2-month-old, exclusively breastfed male infant who had received vitamin K prophylaxis at birth and developed gross hematuria on hospital day 9 while recovering from rotavirus gastroenteritis and cefotaxime-treated aspiration pneumonia. His prothrombin time was 136.9 s (INR 12.6), and activated partial thromboplastin time was 71.1 s. Factors II, VII, IX, and X were each <10%. Urine culture was sterile, and computed tomography demonstrated a right vesicoureteric calculus. A single 1-mg dose of parenteral vitamin K normalized coagulation parameters and resolved the hematuria within four hours. Rotavirus gastroenteritis and prolonged antibiotic use collectively depleted vitamin K stores in this infant despite prophylaxis. VKDB should be considered, particularly when potential risk factors for vitamin K depletion are recognized despite an atypical presentation, and vitamin K administration can be life-saving.
INTRODUCTION:The functional integrity of stored whole blood (WB) is critical for maintaining hemostatic potential during transfusion. While storage-related changes in coagulation components are known, comparative evaluation using viscoelastic and sonorheometric platforms remains limited. This in vitro study explores time-dependent changes in clotting dynamics during WB storage using rotational thromboelastometry (ROTEM) and sonic estimation of elasticity via resonance (SEER) sonorheometry (Quantra). METHODS:WB from eight healthy donors was stored at 1-6 °C in citrate-phosphate-dextrose anticoagulant and sampled on Days 0, 1, 3, 7, 14, and 21. ROTEM parameters included INTEM/EXTEM/FIBTEM clotting time and amplitude; Quantra parameters included clotting time (CT), total clot stiffness (CS), platelet contribution (PCS), and fibrinogen contribution (FCS). Nonparametric statistics and effect sizes were used to assess temporal changes and agreement between platforms. RESULTS:In this pilot in-vitro model ( n = 8) we observed progressive prolongation of clotting time and reduction in clot stiffness were observed across both platforms, as early as 1-3 days into storage. PCS declined significantly, while FCS showed modest reductions. ROTEM and Quantra demonstrated strong correlations in matched parameters, with consistent fixed biases noted in Bland-Altman analysis and a proportional bias with fibrinogen between the two methods. DISCUSSION:Our experimental in vitro study of stored WB exhibits steady, time-dependent hemostatic deterioration, particularly in platelet function and clot initiation. These in-vitro observations reveal early declines in platelet-driven clot stiffness in our model and are hypothesis-generating for further laboratory and clinical evaluation.
OBJECTIVE:Uncontrolled hemorrhage with hemostatic derangement still represents a major preventable cause of death following severe traumatic injury. The purpose of this experimental study was to further investigate the overall hemostasis potential and clot formation after severe traumatic hemorrhage. METHODS:Male New Zealand white rabbits ( n = 12) were subjected to traumatic injury with 40% hemorrhage of their estimated blood volume. The rate of thrombin generation was measured by calibrated automated thrombogram assay, overall hemostatic potential (OHP) by fibrin-aggregation on spectrophotometer, and microvesicles using flow cytometry. The clot structure was analyzed using scanning electron microscopy (SEM). RESULTS:After traumatic hemorrhage, standard laboratory coagulation parameters showed decreased platelet count and fibrinogen, and increased INR and lactate. The OHP and overall coagulation potential (OCP) declined by 59% ( P < 0.001) and 51% ( P < 0,001), respectively. Overall fibrinolytic potential increased from 82% to 86% ( P = 0.008). Platelet derived CD9 positive microvesicles decreased by 84% ( P = 0.002). Endogenous thrombin potential decreased from 483 RFU/min to 383 RFU/min ( P = 0.005). SEM analysis of the fibrin clot structure showed a more porous clot with a decrease in fiber diameter from 72 μm to 56 μm ( P = 0.002). CONCLUSIONS:This experimental study provides important aspects on coagulopathy and clot structure after traumatic hemorrhage including reduced thrombin generation and hyperfibrinolysis resulting in a more porous fibrin clot with lower density and thinner fibers. The methods used may be applied in the clinical research setting to gain further insights about hemostasis and clot formation for targeted therapy after traumatic hemorrhage.
This study aimed to systematically evaluate the antiplatelet activity of dihydromyricetin (DHM) and comprehensively investigate its effects on coagulation function within its therapeutically relevant concentration range. Using in-vitro experiments, we employed a panel of standard assays to assess the effects of DHM on platelet activation and aggregation induced by various agonists [adenosine diphosphate (ADP), arachidonic acid (AA), and collagen (COL)], as well as on agonist-independent adhesion and aggregation. Simultaneously, multidimensional techniques, including conventional coagulation tests, thromboelastography (TEG), and whole-blood dynamic coagulation analysis, were used to systematically evaluate the impact of DHM on the coagulation cascade and overall hemostatic dynamics. The results showed that DHM significantly inhibited platelet activation and aggregation in a concentration-dependent manner and effectively prevented their spontaneous adhesion and aggregation. Crucially, within the concentration range that exerted effective antiplatelet effects, DHM did not significantly affect any of the tested coagulation parameters, and no cytotoxicity was observed. These in-vitro findings suggest that DHM is a platelet-specific inhibitor, exerting potent antiplatelet effects while not interfering with normal physiological coagulation processes. Therefore, DHM shows potential as an antithrombotic candidate pending in-vivo studies with a potentially low risk of bleeding, providing crucial experimental evidence and a new direction for the development of safer antithrombotic strategies.
Uncontrolled bleeding remains a leading cause of preventable death in trauma, and military medicine, underscoring the critical need for effective, accessible available hemostatic agents. While conventional platelet transfusions are indispensable for promoting primary hemostasis, they present several limitations, including short shelf life, logistical challenges, and transfusion-related risks. This review of evidence was done to obtain state-of-the-art data in this field. Hence, this scoping review pays to nonsynthetic platelet-like products, such as platelet microparticles and lyophilized platelet derivatives, which have emerged with promising hemostatic properties. Platelet microparticles, derived from activated platelets, exhibit potent procoagulant activity by contributing to both intrinsic and extrinsic coagulation pathways. Lyophilized platelet products, exemplified by thrombosomes, offer extended shelf life, room-temperature stability, and rapid hemostatic action, making them particularly valuable in prehospital and austere environments. Platelet lysate and PRF, while predominantly explored in regenerative medicine, have under-investigated potential as topical hemostatic agents. Despite encouraging preclinical and early clinical data for some of these products, challenges remain in terms of scalability, regulatory approval, and standardized clinical evaluation. Further research, particularly into platelet lysate and PRF, is warranted to validate their role in hemorrhage control. Nonsynthetic platelet-like products may represent a transformative approach in the management of life-threatening bleeding.
OBJECTIVE:Coagulation tests like prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, international normalized ratio (INR), and D-dimer are vital for monitoring anticoagulant therapy and diagnosing blood disorders. Preanalytical factors such as lipemia, caused by high blood lipid levels after eating, lipid infusions, or metabolic diseases like diabetes, can affect test accuracy. In emergency departments, nonfasting samples increase lipemia risk. This study investigates how lipemia influences coagulation test results to improve diagnostic reliability in clinical settings. MATERIAL AND METHOD:Serial dilutions of 10% Oliclinomel parenteral lipid emulsion were prepared, and 1 ml of each dilution was mixed with plasma samples from 36 patients. The plasma pool was analyzed for PT, aPTT, fibrinogen, INR, and D-dimer using a Sysmex CS2500 analyzer, with triglyceride (TG) concentrations and lipemia index (LI) values measured by a Roche Cobas 6000 C501 analyzer. Bias (%) values were calculated and compared to target bias (%) values to determine cut-off LI values for each parameter. RESULTS:Results showed that PT and fibrinogen were affected by higher TG concentrations, while aPTT was influenced by lower TG concentrations. The cut-off LI values for PT, aPTT, and fibrinogen were determined to be 300, 50, and 150, respectively. For D-dimer, the cut-off LI value was established at 1200. CONCLUSION:These findings highlight the importance of determining specific LI cut-off values for each laboratory and considering lipemia's effect on coagulation test results, particularly in emergency settings and in patients with dyslipidemia.
Congenital fibrinogen disorders are rare. We describe a novel fibrinogen γ-chain variant and review adjacent variants to contextualize its clinical significance. A woman investigated for infertility and her mother underwent hemostatic evaluation including functional, antigenic fibrinogen, and fibrin turbidity assays. Whole-exome sequencing (WES) was performed. Both asymptomatic individuals had hypodysfibrinogenemia with disproportionately reduced functional and antigenic fibrinogen and impaired fibrin polymerization with prolonged lag phase and reduced maximal slope (Vmax). WES revealed a novel heterozygous FGG missense variant, c.1172A>T (p.Asn391Ile; γ[365]Asn>Ile), in both individuals. According to ISTH-SSC guideline, both met subtype 4C (mild hypodysfibrinogenemia). Across 11 published cases with variants in γ361-369, six showed impaired fibrin polymerization and phenotypes ranged from bleeding ( n = 6) and venous thrombosis ( n = 2) to asymptomatic ( n = 2) and hepatic fibrinogen storage disease ( n = 1). FGG p.Asn391Ile variant is associated with hypodysfibrinogenemia and impaired fibrin polymerization, but current evidence is insufficient to define its clinical phenotype.
INTRODUCTION:Constitutional thrombopathies, also known as inherited platelet disorders (IPDs), represent a complex and heterogeneous group of bleeding disorders. These conditions are still poorly documented in resource-limited settings, particularly in sub-Saharan Africa. This study was initiated with the primary objective of confirming suspected cases of IPD in Senegal. METHODOLOGY:We conducted an observational study of all suspected cases of constitutional thrombopathy at the clinical hematology department in Dakar. Each patient provided written consent, and complete clinical files were obtained. We analyzed clinical parameters such as bleeding antecedent, history of care, first symptoms, bleeding profile, ISTH-SSC Bleeding Assessment Tool (BAT), and any complications related to the disorder.Biological assessments included platelet counts, blood smears, PT, aPTT, fibrinogen levels, coagulation factor assays, platelet aggregation testing using five agonists (collagen, ADP, AA, epinephrine, and ristocetin), and platelet immunophenotyping by flow cytometry (FC). RESULTS:We identified ten cases, of which seven were included in this study. All patients were from consanguineous marriages, and only two had no family history of bleeding. Clinical manifestations were predominantly mucosal hemorrhages. All patients had elevated ISTH-SSC BAT.Platelet aggregation and immunophenotyping confirmed a Bernard-Soulier syndrome profile in one patient and a Glanzmann thrombasthenia in four patients. The remaining two patients exhibited profiles suggestive of GPVI/α2β1 integrin and P2Y1/P2Y12 receptor deficiencies. CONCLUSION:This first study conducted locally in sub-Saharan Africa highlights the complexity of diagnosing inherited platelet disorders and the challenges of implementing light transmission aggregometry and platelet immunophenotyping in resource-limited countries.
This study assessed the effect of low-dose aspirin (81 mg) on thrombin generation in healthy volunteers using the calibrated automated thrombogram (CAT). This single-center prospective crossover study involved healthy volunteers, each serving as their own control. After Institutional Review Board approval and informed consent, healthy participants were administered aspirin (81 mg daily) for 7 days. Blood samples were collected at baseline, after the 7-day aspirin regimen, and 2 weeks postaspirin discontinuation. Platelet-poor plasma (PPP) samples were analyzed using CAT to measure endogenous thrombin potential (ETP) and other CAT parameters. The primary endpoint was change in ETP from baseline to after 7 days of aspirin. Data were analyzed with paired t tests. Data from 18 participants (ages 27-54) showed no significant change in ETP after aspirin administration (primary endpoint). Specifically, mean (standard deviation) ETP at baseline and on-aspirin were 1750 (237) vs. 1832 (301) nmol/l min, respectively. Secondary CAT parameters (lag time, peak thrombin generation, time to peak, and velocity index) also showed no significant differences. We did not observe a significant effect of low-dose aspirin (81 mg) for 7 days on any measure of thrombin generation in healthy volunteers. This study's controlled design in healthy subjects contrasts with previous studies that focused on patients where confounding effects of comorbidities and concomitant medications may have influenced the results.
The clearance of rivaroxaban and apixaban is mediated via cytochrome P450 3A4 (CYP3A4) and Permeability-glycoprotein (P-gp) at varying extents. Amiodarone, a weak CYP3A4 and moderate P-gp inhibitor, has the potential to reduce the clearance of these factor Xa (FXa) inhibitors. The real-world impact of this drug-drug interaction is unclear. Although international normalised ratio (INR) is not an accurate measure of FXa inhibitor effect, changes in INR may indicate changes in the impact of FXa inhibitors. To determine what effect the addition of amiodarone has on INR in patients concurrently prescribed rivaroxaban or apixaban in clinical practice. This retrospective observational study identified patients with atrial fibrillation prescribed rivaroxaban or apixaban from April 2017 to December 2022. INRs were analysed in patients prescribed rivaroxaban or apixaban with or without concomitant amiodarone. One hundred and forty-seven patients were included in the study and were separated into the following cohorts: patients prescribed apixaban alone, rivaroxaban alone, apixaban with amiodarone, or rivaroxaban with amiodarone. Mean INRs were greater for patients prescribed apixaban with amiodarone compared to those prescribed apixaban only (1.56 vs. 1.29, respectively) but not for patients prescribed rivaroxaban with amiodarone compared to patients prescribed rivaroxaban only (1.57 vs. 1.61, respectively). Four patients prescribed apixaban with amiodarone had INRs greater than or equal to 3. Our real-world data support literature suggesting that the amiodarone apixaban interaction is real and that there is a large inter-individual variability in the magnitude of this interaction. Further research analysing other coagulation markers (i.e. anti-Xa activity) is warranted to identify at-risk cohorts.
Background We have previously shown that factor XIII (FXIII) levels should be above 30% in an acute care setting. The aim of this analysis was to assess morbidity and mortality in a larger sample of patients with acquired FXIII deficiency, and to explore the potential underlying causes of acquired FXIII deficiency. Material and methods We conducted a retrospective analysis of all patients diagnosed with acquired factor XIII deficiency at a large hospital over a period of 24 months (study ID: NCT04416594; http://www.clinicaltrials.gov). We then assessed standardized clinical and outcome data. Results We included 65 patients with FXIII activity below 70%, and a subsequent analysis included a further 104 patients (including those from our previous report). FXIII activity was significantly lower in patients who massively bled and in those admitted to the intensive care unit (ICU). Low FXIII activity was significantly associated with longer ICU stays, a high incidence of major bleeding and multiple organ dysfunction syndrome (MODS), and increased 1- and 2-year mortality. However, we found no evidence of deranged FXIII activation in this acute care setting. Conclusions Acquired factor XIII deficiency is associated with high morbidity and mortality rates. In the setting of acquired FXIII deficiency, the level of FXIII required for haemostasis and endothelium stabilization seem to be around 40%. Activation of FXIII seems to be warranted in order to stop bleeding.
In this study, we aimed to evaluate the precision, accuracy, linearity, reference range, hemolysis-lipemia interactions, and carryover of coagulation tests [prothrombin time (PT), activated partial prothrombin time (aPTT), fibrinogen (Fib), and D-dimer]. Precision was assessed by running normal and pathological control materials. For method comparison, 41 patients' samples were analyzed on both Sysmex-CS-2500 (optical) and STA-Compact (mechanical) analyzers. The results were evaluated using Passing-Bablock regression and Bland-Altman plots. Linearity and carry-over were performed by serial dilution of high and low-concentration samples and alternating normal and pathological samples. Interference from hemolysis and lipemia was investigated by creating plasma pools with varying hemoglobin and lipid levels and comparing the results with the manufacturer's thresholds.
Interactions between medicinal plants and conventional drugs represent a growing public health concern, particularly with antithrombotic medications due to increased bleeding risk. This study aimed to evaluate in vitro the antioxidant and antithrombotic properties of Tetraclinis articulata aqueous extract. Leaves were collected in Oujda, dried, crushed, and extracted by aqueous infusion. Total phenolic content was measured using the Folin–Ciocalteu method. Antioxidant activity was assessed via DPPH and ABTS assays. Antithrombotic activity was evaluated using blood from healthy donors. Platelet aggregation was measured with optical aggregometry using thrombin as the agonist. Anticoagulant effects were assessed through prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen concentration. Fibrinolytic activity was determined by a clot-lysis assay. The extract contained 70.55 μg GAE/mg of phenolics and showed strong antioxidant activity (IC50 = 20.26 ± 0.54 μg/ml for DPPH and 8.56 ± 0.93 μg/ml for ABTS). It inhibited platelet aggregation in a dose-dependent manner: 35.2% at 0.5 mg/mL, 64.1% at 1 mg/ml, and 92.18% at 2 mg/ml. The extract significantly prolonged PT and aPTT and reduced fibrinogen levels. Clot lysis reached 34.6% at 1 mg/ml. Tetraclinis articulata exhibited potent antioxidant, antiaggregant, anticoagulant, and fibrinolytic activities in vitro . These findings support its potential as a natural antithrombotic agent. However, its strong antiaggregant effect warrants caution regarding potential adverse interactions when used alongside conventional antithrombotic treatments.
Romiplostim is a thrombopoietin receptor agonist indicated for previously treated primary immune thrombocytopenia (ITP) that leads to dose-dependent increases in platelet counts. Higher initial doses than the standard starting dose of 1 μg/kg may have the potential to lead to faster platelet responses. We conducted a single-center, retrospective, observational study to evaluate the efficacy of romiplostim dosing strategies in adult patients with primary ITP who initiated romiplostim in the acute care setting. Patients were classified into low-dose (<3 μg/kg) or high-dose (≥3 μg/kg) based on the initial weight-based dose administered. The primary outcome was time to achieve a platelet count greater than 30 × 10 9 /l. Secondary objectives included time to platelet count greater than 50 × 10 9 /l, healthcare resource utilization, thrombotic events, and bleeding incidents. Sixty-five patients were included in the study. Patients in both dosing cohorts achieved a platelet response of greater than 30 × 10 9 /l at a median of 7 days ( P = 0.871). There were no significant differences in healthcare resource utilization or safety events between dosing cohorts. Initial high-dose romiplostim for acute ITP did not demonstrate an improvement in time to platelet response compared to initial low-dose romiplostim.
Persons with bleeding disorders (PwBD) often face delays in receiving hemostatic therapies within the recommended 60-min window upon presenting to the emergency department, potentially exacerbating their conditions. We aimed to investigate the emergency department workflow for managing PwBD to identify inefficiencies and barriers to timely treatment. We conducted a retrospective cohort study of patients with hemophilia and von Willebrand disease (VWD) presenting to the emergency department from 2019 to 2022. Data were collected on patient demographics, disease type, severity, treatment type, and prescriber specialty. Time from triage to medication administration was the primary outcome. Statistical comparisons and Firth's penalized logistic regression were performed. Among 241 emergency department visits in 122 PwBD, 50% of the 86 bleeding events were treated with hemostatic therapies. Only 11% received them within 60 min. The median time from emergency department triage to administration of hemostatic therapies was 175 min [interquartile range (IQR) 93–364] for clotting factor concentrates (CFCs) and 206 min (IQR 152–269) for desmopressin. Important delays were observed in patient rooming, physician initial assessment, ordering and administration of hemostatic therapies. Patients triaged more urgently may have experienced higher odds of receiving hemostatic therapies in 60 min or less, though regression analysis did not identify statistically significant predictors of treatment beyond 60 min. This study highlights the urgent need for targeted interventions to address systemic challenges and mitigate treatment delays for PwBD in Canadian emergency department settings. Future research should focus on prospective evaluations of intervention effectiveness and the role of patient self-management in reducing emergency department dependency.
