Explore the vast range of titles available.

Thrombin generation (TG) assays evaluate the balance between pro‐ and anticoagulant forces, to better assess bleeding and thrombotic risks. Although TG readouts obtained with the calibrated automated TG have been investigated in multiple clinical conditions, TG still needs standardization and clinical validation. The automated TG instrument ST Genesia® (STG, Stago, Asnières‐sur‐Seine, France) provides a normalization of TG parameters based on a reference plasma aiming to reduce the interlaboratory variability and the variability between different measurement runs. To evaluate STG in a group of healthy adults. Reference intervals in healthy adults and variability of the new standardized reagents for bleeding (BleedScreen) and thrombophilic (ThromboScreen) conditions were determined using STG. TG was measured in platelet‐free plasma (PFP) samples of 123 healthy adults. Reference intervals were determined for TG parameters. Intra‐ and interassay coefficients of variation were calculated on quality controls and PFP samples from healthy adults. Oral contraception (OC) possibly influenced TG parameters, resulting in a higher median and a broader reference interval for peak height and endogenous thrombin potential (ETP) in women aged 20 to 49 years than in all other sex and age categories. Therefore, we propose the following reference interval categories: men, women aged <50 years not using OC, women aged <50 years using OC, and women aged ≥50 years. Normalization was effective to reduce the interassay variability of quality controls for ETP (BleedScreen assay), and peak height and ETP (ThromboScreen assay without thrombomodulin), but had little impact on PFP sample variability. STG appears suitable for accurate measurement of TG in healthy adults.

The ability to assess the hemostatic effect of the direct oral anticoagulant (DOACs) may be valuable in clinical situations such as bleeding or thrombosis, before urgent surgery, or reversal of anticoagulation. We sought to assess the anticoagulant effect of DOACs with the new‐generation fully automated thrombelastograph TEG 6s using resonance‐frequency viscoelasticity measurements and disposable multichannel microfluidic cartridges. A single dose of oral dabigatran 150 mg, rivaroxaban 20 mg, or apixaban 5 mg was given to 9 healthy males. Phlebotomy was performed at 0, 1, and 3 hours after administration of DOAC. TEG parameters were measured using TEG_6s. Concentrations of DOACs were measured using chromogenic assays. The TEG parameters were correlated to the DOAC concentrations. The reaction time (R) demonstrated the strongest response to DOAC intake. There were no correlations between other TEG parameters and DOAC concentrations. Using the direct thrombin inhibitor (DTI) channel, R was significantly correlated with dabigatran levels (r=0.94, P<0.0001). Using the anti–factor Xa (AFXa) channel, R was significantly correlated with rivaroxaban and apixaban levels (r=0.93 and r=0.83, respectively; P<0.0001 for both). R >2.5 minutes for dabigatran (DTI channel), >2.5 minutes for apixaban, and >1.8 minutes for rivaroxaban (AFXa channel) were associated with 100% sensitivity and ≥ 90% specificity to detect DOAC levels of ≥ 50 ng/mL. We have demonstrated that TEG_6s R has significant correlation with DOAC blood concentrations and has potential for monitoring the DOAC's effect on hemostasis with reasonable sensitivity in the small sample analyzed. This novel technology is easy to use on a small volume of whole blood without requiring a specialized laboratory. Further study is warranted to correlate R with clinical outcomes.

Current treatment for severe hemophilia A is replacement of deficient factor. Although replacement therapy has improved life expectancy and quality, limitations include frequent infusions and high costs. Gene therapy is a potential alternative that utilizes an adeno‐associated virus (AAV) vector containing the human genetic code for factor 8 (FVIII) that transduces the liver, enabling endogenous production of FVIII. Individuals with preexisting immunity to AAV serotypes may be less likely to benefit from this treatment. This study measured seroprevalence of antibodies to AAV5 and 8 in an UK adult hemophilia A cohort. Patients were recruited from seven hemophilia centres in the UK. Citrated plasma samples from 100 patients were tested for preexisting activities against AAV5 and 8 using AAV transduction inhibition and total antibodies assays. Twent‐one percent of patients had antibodies against AAV5 and 23% had antibodies against AAV8. Twenty‐five percent and 38% of patients exhibited inhibitors of AAV5 or AAV8 cellular transduction respectively. Overall seroprevalence using either assay against AAV5 was 30% and against AAV8 was 40% in this cohort of hemophilia A patients. Seropositivity for both AAV5 and AAV8 was seen in 24% of participants. Screening for preexisting immunity may be important in identifying patients most likely to benefit from gene therapy. Clinical studies may be needed to evaluate the impact of preexisting immunity on the safety and efficacy of AAV mediated gene therapy.

A prolonged activated partial thromboplastin time (APTT) of unknown cause is one of the most frequent reasons why outpatients are referred for hemostasis consultation. Nevertheless, very few data are available on the relative contribution of individual causes of this common clinical scenario. Here, we present a systematic evaluation of all causes of APTT prolongation in a consecutive population of outpatients referred for specialized hemostasis consultation during a 14‐year period. All cases referred to an academic specialized hemostasis outpatient unit due to APTT prolongation of unknown etiology whose prolonged APTT was confirmed in the first visit were included in the study. Data were obtained from the electronic medical records. Among 187 consecutive patients, the most frequent causes were antiphospholipid antibodies in 22.6%, contact pathway factor deficiencies in 17.4%, other coagulation factor deficiencies in 11.6%, and vitamin K deficiency/liver disease in 11.6%. A definite cause was not identified in 22.1% of patients. Presence of antiphospholipid antibodies, and absence of bleeding symptoms were both associated with significantly longer APTT values compared to other categories/clinical scenarios. The investigation of each case required a mean of 18.2 additional tests per patient, with estimated costs ranging from US$191.60 to US$1055.60. Our results describe the main causes of APTT prolongation in outpatients, as well as estimates of resource use required to investigate this condition, thus providing evidence supporting the importance of measures to minimize the indiscriminate use of this assay.

Unlabelled Box Fibrinogen is a complex molecule comprised of two sets of Aα, Bβ, and γ chains. Fibrinogen deficiencies can lead to the development of bleeding or thromboembolic events. The objective of this study was to perform DNA sequence analysis of patients with clinical fibrinogen abnormalities, and to perform genotype‐phenotype correlations. DNA from 31 patients was sequenced to evaluate disease‐causing mutations in the three fibrinogen genes: FGA, FGB, and FGG. Clinical data were extracted from medical records or from consultation with referring hematologists. Fibrinogen antigen and functional (Clauss method) assays, as well as reptilase time (RT) and thrombin time (TT) were obtained for each patient. Molecular modeling was used to simulate the functional impact of specific missense variants on the overall protein structure. Seventeen mutations, including six novel mutations, were identified in the three fibrinogen genes. There was little correlation between genotype and phenotype. Molecular modeling predicted a substantial conformational change for a novel variant, FGG p.Ala289Asp, leading to a more rigid molecule in a region critical for polymerization and alignment of the fibrin monomers. This mutation is associated with both bleeding and clotting in the two affected individuals. Robust genotype‐phenotype correlations are difficult to establish for fibrinogen disorders. Molecular modeling might represent a valuable tool for understanding the function of certain missense fibrinogen mutations but those should be followed by functional studies. It is likely that genetic and environmental modifiers account for the incomplete penetrance and variable expressivity that characterize fibrinogen disorders.

Although flow cytometry is often brought forward as a preferable method in the setting of thrombocytopenia, the relative effects of low sample counts on results from flow cytometry‐based platelet function testing (FC‐PFT) in comparison with light transmission aggregometry (LTA) and multiple electrode aggregometry (MEA) has not been reported. To compare the effects of different sample platelet counts (10, 50, 100, and 200 × 109 L−1) on platelet activation measured with FC‐PFT, LTA, and MEA using the same anticoagulant and agonist concentrations as for the commercial MEA test. Platelets were stimulated with two commonly used platelet agonists (ADP [6.5 μmol L−1] and PAR1‐AP [TRAP, 32 μmol L−1]). The specified sample platelet counts were obtained by combining platelet‐rich and platelet poor hirudinized plasma in different proportions with or without red blood cells. For FC, P‐selectin exposure and PAC‐1 binding was reduced at 10 × 109 L−1 after stimulation with PAR1‐AP (by approximately 20% and 50%, respectively), but remained relatively unchanged when ADP was used as agonist (n = 9). The platelet count‐dependent effects observed with PAR1‐AP were eliminated when samples were pre‐incubated with apyrase, implying that reduced purinergic signaling was the main underlying factor (n = 5). Both aggregometry‐based PFTs showed a 50% reduction at 50 × 109 L−1 and more than 80% reduction at 10 × 109 L−1, irrespective of agonist used (n = 7). Although FC‐PFT is generally preferable to aggregometry‐based PFTs in situations with low sample platelet counts, a careful optimization of experimental parameters is still required in order to eliminate platelet count‐related effects.

The phenotypic diagnosis of von Willebrand disease (VWD) is a multistep process with classification dependent on the quantification of von Willebrand factor (VWF) multimeric structure. VWF multimer analysis is a technically challenging, lengthy and non‐standardised assay, usually performed in specialist laboratories. Recently, a new semi‐automated multimer assay, the Hydragel 5 von Willebrand multimers (H5VWM) has become available. This study, performed in two European centres, compared existing in‐house multimer assays to the H5VWM in individuals with and without VWD. Overall agreement of 91.1% was observed in 74 individuals with normal VWF levels, 57 patients grouped as type 1 VWD, 33 type 2A, 16 type 2B, 28 type 2M, 11 type 2N. Patients tested following Desmopressin or VWF concentrate, with thrombotic thrombocytopenic purpura and acquired von Willebrand syndrome were also evaluated. Many of the discrepancies between methods were in patients with genetic mutations linked to more than one type of VWD including p.R1374C/H and p.R1315C. Quantifiable multimer results were available within one working day. Densitometry improved the interpretation of the multimers with slight structural variations that were not apparent by visual inspection of the in‐house method. 5VWM was a rapid, sensitive, standardised assay which used existing technology and could be included as an initial screen of VWF multimers in a VWD diagnostic algorithm in conjunction with traditional multimer analysis.

Investigations of platelet function by light transmission aggregometry (LTA) using a dedicated aggregometer is time consuming and labor intensive. This multicenter study evaluated an automated LTA method using a coagulation analyzer to establish reference ranges and ideal testing regimen. Sysmex CS‐2x00 series analyzers were used to measure aggregation using a range of agonists and concentrations: ADP (1‐20 μM); arachidonic acid (0.5‐1.5 mM); collagen (1.25‐5 μg/mL); ristocetin (0.5‐1.5 g/L); epinephrine (5‐10 μM); TRAP (1‐20 μM); U46619 (1 μM); and saline. Maximum and final aggregation, disaggregation, slope, and acquisition time were compared for each. For 42 normal subjects there was no significant difference in aggregation parameters for: 10 μM and 20 μm ADP; 2 and 2.5 μM ADP; 1 and 1.5 mM arachidonic acid; 2.5 and 5 μg/mL collagen; 1 and 1.25 μg/mL collagen; 1.25 and 1.5 g/L ristocetin; 5 and 10 μM epinephrine; 5 and 10 μM or 20 μM TRAP. Maximum aggregation was reached by 300 seconds with 20 and 10 μM ADP, 1 μM U46619, 1 and 1.25 μg/mL collagen, 1.5 g/L ristocetin and 5, 10, and 20 μM TRAP: all others agonists required 600s. A standard panel of agonists can be used on the Sysmex CS‐2x00 series analyzers: ADP (10, 5, 2.5, and 1.25 μM); 1 mM arachidonic acid; 1 μM U46619; 2.5 and 1.25 μg/mL collagen; 1.25 and 0.5 g/L ristocetin; 5 μM epinephrine; 5 and 10 μM TRAP; and saline. Aggregation should be observed for 600 seconds for all agonists except TRAP and U46619, which require 300 seconds. If further studies confirm these concentrations detect platelet disorders then Sysmex CS‐series analyzers could replace dedicated aggregometers, or perform LTA where it is currently not available.

Unlabelled Box FV‐Short is a normal splice variant of Factor V (FV) having a short B domain, which exposes a high affinity‐binding site for tissue factor pathway inhibitor α (TFPIα). FV‐Short and TFPIα circulate in complex in plasma. The aim was to elucidate whether FV‐Short affects TFPIα as inhibitor of coagulation FXa and to test whether the TFPIα‐cofactor activity of protein S is influenced by FV‐Short. Recombinant FV, wild‐type FV‐Short and a FV‐Short thrombin‐cleavage resistant variant were expressed and purified. The influence of FV and FV‐Short variants and/or protein S on the FXa inhibitory activity of TFPIα was monitored both in a purified system and in a plasma‐based thrombin generation assay. FV‐Short had intrinsically weak TFPIα‐cofactor activity but with protein S present, FV‐Short yielded efficient inactivation of FXa. Protein S alone did not promote full TFPIα‐activity. Intact FV was inefficient at low protein S concentrations and had 10‐fold lower activity compared to FV‐Short at physiological protein S levels. Activation of FV‐Short by thrombin resulted in the loss of the TFPIα‐cofactor activity. The synergistic TFPIα‐cofactor activity of FV‐Short and protein S was also demonstrated in plasma using a thrombin generation assay. FV‐Short and protein S are highly efficient, synergistic cofactors to TFPIα in the regulation of FXa activity, whereas full length FV has lower activity. Our results suggest the formation of an efficient FXa‐inhibitory complex between FV‐Short, TFPIα and protein S on the surface of negatively charged phospholipids.

Unlabelled Box Factor VIII (FVIII) replacement is standard of care for patients with hemophilia A (HemA); however, patient response does not always correlate with FVIII levels. We hypothesize this may be in part due to the physical properties of clots and contributions of fibrin, platelets, and erythrocytes, which may be important for hemostasis. To understand how FVIII contributes to effective hemostasis in terms of clot structure and mechanical properties In vitro HemA clots in human plasma or whole blood were analyzed using turbidity waveform analysis, confocal microscopy, and rheometry with or without added FVIII. In vivo clots from saphenous vein puncture in wild‐type and HemA mice with varying FVIII levels were examined using scanning electron microscopy. FVIII profoundly affected HemA clot structure and physical properties; added FVIII converted the open and porous fibrin meshwork and low stiffness of HemA clots to a highly branched and dense meshwork with higher stiffness. Platelets and erythrocytes incorporated into clots modulated clot properties. The clots formed in the mouse saphenous vein model contained variable amounts of compressed erythrocytes (polyhedrocytes), fibrin, and platelets depending on the levels of FVIII, correlating with bleeding times. FVIII effects on clot characteristics were dose‐dependent and reached a maximum at ~25% FVIII, such that HemA clots formed with this level of FVIII resembled clots from unaffected controls. Effective clot formation can be achieved in HemA by replacement therapy, which alters the architecture of the fibrin network and associated cells, thus increasing clot stiffness and decreasing clot permeability.