scholarly journals Evaluation of Global Coagulation Tests for Monitoring Bleeding Phenotypes and Response to Treatments in FVII Deficiency

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1046-1046
Author(s):  
Elena G Arias-Salgado ◽  
María Isabel Rivas Pollmar ◽  
Elena Monzón Manzano ◽  
Paula Acuña ◽  
María Teresa Alvarez Román ◽  
...  
Keyword(s):  
Research Funding ◽  
Thrombin Generation ◽  
Thrombus Formation ◽  
Severe Bleeding ◽  
High Shear ◽  
Clotting Time ◽  
Blood Samples ◽  
Replacement Treatment ◽  
Coagulation Tests ◽  
Fvii Deficiency

Abstract Introduction: The management of Factor (F) VII deficiency is complex due to the lack of a clear correlation between FVII levels and the bleeding manifestations of the patients, with a variety of responses to the treatments. Additionally,the presence of FVII inhibitors may also occur. Thus, it is essential to be able to monitoring the hemostatic profile and the effect of the different therapies in each patient individually. Our aim was to perform a personalized analysis of the coagulation status of patients with FVII deficiency and to evaluate their response to new potential therapies using five different coagulation tests. Methods: Six patients were included (clinical data in Table-1):4 with bleeding symptoms on prophylaxis with recombinant activated FVII (rFVIIa), one of them with FVII inhibitors; and 2 untreated patients without bleeding episodes. Blood samples obtained from patients on prophylaxis were collected predose. Prothrombine time (PT) and activated partial thromboplastin time (aPTT)were tested using HemosIL ® RecombiPlasTin 2G, and SynthASil respectively. Rotational Thromboelastometry (ROTEM ®) was performed for monitoring the clot formation using blood with corn trypsin inhibitor (CTI) to inhibit contact activation and a low amount of Tissue Factor (TF) as trigger (dilution 1:50,000 of EXTEM reagent). The thrombin generation (TG) was tested with the Calibrated Automated Thrombogram (CAT) system using platelet poor plasma (PPP) obtained from blood with CTI and activated with only 1 pM TF plus phospholipids (PPP-Low ®, Stago). The plasmin generation (PG) was measured in citrated PPP using a comercial kit (Synapse). The total thrombus-formation analysis system (T-TAS ®, Zacros) was conducted by loading CTI blood samples in AR-chips coated with collagen and thromboplastin for assessing thrombus formation mediated by the activation of the coagulation under flow conditions (High shear). The Area under the flow pressure curve (AUC) was calculated over 30 min after starting. Effects of ex vivo spiking doses of factor replacement (rFVIIa) or non-factor replacement treatments (anti-TFPI, clone mAb2021, Creative Biolabs) were tested. Results: aPTT values remained normal in all patients. FVII deficiency significantly affected PT and patients with more severe bleeding phenotype (patient #1, 2, 3, and 4) showed much longer PT values. Similarly, ROTEM and T-TAS assays showed that FVII deficiency only caused an important delayed clotting time (CT) and very anomalous thrombus formation (AUC) in patients with severe bleeding symptoms.More prolonged lag time (LT) and an important decrease in the peak of thrombin and plasmin generation was also observed in the same patients (#1, 2, 3, and 4). Patient #1 with FVII inhibitors presented a more affected hemostatic profile according to all the coagulation parameters obtained with the five different assays. In contrast, the patients #4 and #5 with absence of bleeding complications showed most of these values within the normal reference range obtained from healthy controls. Concentrations of 1µg/ml (equivalent to 90 μg/kg) of the factor-replacement treatment rFVIIa, showed the normalization of the PT, the clotting time (CT), and the restoration of the thrombin and plasmin generation, and the regularization of the coagulation-dependent thrombus formation in all the patients. In vitro spiking with anti-TFPI (400-800 ng/ml), an alternative non-factor replacement treatment,corrected the thrombus formation (AUC) defects under high shear flow observed in the patients, and produced a significant reduction of CT and LT, and increments of thrombin generation although less effectively than the factor replacement therapy. Conclusions: All the global tests, performed with the described conditions in this study, were sensitive enough to show an abnormal hemostatic profile in the FVII-deficient patients with worst clinical symptoms, validating their use to monitor the risk of bleeding events and the responses to different treatments in this deficiency. These assays may allow to monitoring more personalized treatments to these patients. The results also pointed to the possibility that inhibition of TFPI might be useful for treatment of patients with FVII deficiency, opening the idea of its usage especially as an alternative therapy for patients with inhibitors. Research funded by ISCIII-Fondos FEDER PI19/00772 and PI19/00631 Figure 1 Figure 1. Disclosures Alvarez Román: Pfizer: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. García Barcenilla: Roche: Speakers Bureau; Takeda: Speakers Bureau; SOBI: Speakers Bureau; Bayer: Speakers Bureau. Canales: Takeda: Consultancy, Honoraria, Speakers Bureau; Karyopharm: Consultancy, Honoraria; Incyte: Consultancy; Sanofi: Consultancy; Novartis: Consultancy, Honoraria; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; iQone: Honoraria; Sandoz: Honoraria, Speakers Bureau; Eusa Pharma: Consultancy, Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Gilead/Kite: Consultancy, Honoraria. Butta: CSL-Behring: Research Funding; Roche: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Novo-Nordisk: Speakers Bureau. Jiménez-Yuste: Octapharma: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Pfizer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding.

scholarly journals Prospective Assessment of Biomarkers of Hypercoagulability in Oncological Patients and Healthcare Workers Following Vaccination Against Sars-Cov-2 with the mRNA Vaccine. the Roadmap-COVID-19-Vaccin Study

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3207-3207
Author(s):  
Patrick Van Dreden ◽  
Joseph Gligorov ◽  
Evangelos Terpos ◽  
Mathieu Jamelot ◽  
Michele Sabbah ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Tissue Factor ◽  
Platelet Activation ◽  
Research Funding ◽  
Thrombin Generation ◽  
Cell Activation ◽  
Control Group ◽  
Clotting Time ◽  
Endothelial Cell Activation ◽  
Active Cancer

Abstract Background: COVID-19 has been associated with hypercoagulability, endothelial cell injury and frequent thrombotic complications resulting both from direct effects of the virus on the endothelium and from the 'cytokine storm' resulting from the host's immune response. Since the COVID-19 vaccines have been shown to effectively prevent symptomatic infection including hospital admissions and severe disease, the risk of COVID-19-related thrombosis should be expected to (almost) disappear in vaccinated individuals. However, some rare cases of venous thrombosis have been reported in individuals vaccinated with mRNA vaccines. Thus, there is a sharp contrast between the clinical or experimental data reported in the literature on COVID-19 and on the rare thrombotic events observed after the vaccination with these vaccines. This phenomenon raised some scepticism of even some fear about the safety of these vaccines which could compromise the adhesion of the citizens in the vaccination program. Aims: We conducted a prospective observational study, to explore the impact of vaccination with the BNT162b2 (Pfizer/BioNTech) on blood hypercoagulability and endothelial cell activation and to investigate if this is modified by the presence of active cancer. Methods: In total 229 subjects were prospectively included in the study from April to June 2021. Subjects were stratified in three predefined groups: 127 vaccinated patients with active cancer (VOnco group), 72 vaccinated health care workers (VHcw group) and 30 non vaccinated health individuals (Control group). Blood samples were obtained 2 days after the administration of the first dose of BNT162b2 vaccine and collected in Vacutainer® tubes (0.109 mol/L trisodium citrate). Platelet poor plasma (PPP) was prepared by double centrifugation at 2000 g for 20 minutes at room temperature and plasma aliquots were stored at -80°C until assayed. Samples of PPP were assessed for thrombin generation (TG) with PPP-Reagent® (Thrombogram-Thrombinoscope assay with PPP-Reagent®TF 5pM), E-selectin, D-dimers, (D-Di), Tissue Factor (TFa), procoagulant phospholipid-dependent clotting time (Procag-PPL) and von Willebrand factor (vWF), thrombomodulin (TM), tissue factor pathway inhibitor (TFPI), and platelet factor 4 (PF4). All assays were from Diagnostica Stago (France). The upper and lower normal limits (UNL and LNL) for each biomarker were calculated by the mean±2SD for the control group. Results: All vaccinated subjects showed significantly increased levels of PF4 (71% >UNL, p<0.001), D-Dimers (74% >UNL, p<0.01), vWF (60% >UNL, p<0.01), FVIII (62% >UNL, p<0.01) and shorter Procoag-PPL clotting time (96% <LNL, p<0.001), as compared to controls. Thrombin generation showed significantly higher Peak (60% >UNL, p<0.01), ETP (38% >UNL, p<0.01) and MRI (66% >UNL, p<0.01) but no differences in lag-time in vaccinated subjects as compared to the control group. Vaccinated subjects did not show any increase at the levels of TFa, TFPI, TM and E-selectin in comparison with the control group. The studied biomarkers were not significantly different between the VOnco and VHcw groups. Conclusion: The ROADMAP-COVID-19-Vaccine study shows that administration of the first dose of the BNT162b2 vaccine induced significant platelet activation documented by shorter Procoag-PPL associated with increased levels of PF4. Plasma hypercoagulability was less frequent in vaccinated individuals whereas there was no evidence of significant endothelial cells activation after vaccination. Interestingly, the presence of active cancer was not associated with an enhancement of platelet activation, hypercoagulability, or endothelial cell activation after the vaccination. Probably, the generated antibodies against the spike protein or lead to platelet activation in a FcyRIIa dependent manner that results in PF4 release. The implication of the mild inflammatory reaction triggered by the vaccination could be another possible pathway leading to platelet activation. Nevertheless, vaccination does not provoke endothelial activation even in patients with cancer. The findings of the ROADMAP-COVID-19-Vaccine study support the concept administration of mRNA based vaccines does not directly cause a systematic hypercoagulability. Disclosures Gligorov: Roche-Genentech: Research Funding; Novartis: Research Funding; Onxeo: Research Funding; Daichi: Research Funding; MSD: Research Funding; Eisai: Research Funding; Genomic Heatlh: Research Funding; Ipsen: Research Funding; Macrogenics: Research Funding; Pfizer: Research Funding. Terpos: Novartis: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Genesis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; GSK: Honoraria, Research Funding. Dimopoulos: Amgen: Honoraria; BMS: Honoraria; Janssen: Honoraria; Beigene: Honoraria; Takeda: Honoraria.

scholarly journals Thrombin Generation Related to Netosis in Patients with Systemic Lupus Erythematosus

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 10-11
Author(s):  
Elena Monzón Manzano ◽  
Ihosvany Fernandez-Bello ◽  
Raul Justo Sanz ◽  
Ángel Robles Marhuenda ◽  
Paula Acuña ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Fluorescence Microscopy ◽  
Lupus Erythematosus ◽  
Antiphospholipid Antibodies ◽  
Research Funding ◽  
Thrombin Generation ◽  
Healthy Controls ◽  
Systemic Lupus ◽  
Blood Samples ◽  
History Of

NETosis is a process suffered by neutrophils that consists in the loss of their function and the release of their nuclear material as large web-like structure called neutrophil extracelular traps (NETs). Many authors demonstrated that NETs participate in the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE), because the release of autoantigens amplifies inflammatory responses, perpetuating the exacerbation of autoimmunity. On the other hand, NETs may play a prominent role in thrombosis because they serve as a negative charge scaffold to trap platelets and coagulation factors, promoting blood clot formation. Objetive: to determine participation of NETs in the hypercoagulable state of patients with SLE. Methods: 32 patients with SLE without antiphospholipid antibodies and without history of thrombotic events were included after signing informed consent; 88 sex- and age-matched healthy controls were also recruited. Blood samples were drawn in citrate tubes (3.2%). Neutrophils were isolated by centrifugation of whole blood with a Percoll gradient at 500 g, 25 min, 5ºC. To induce NETs formation, 2.5x105 isolated neutrophils were incubated in RPMI-1640 medium with or without 100 nM phorbol 12-myristate 13-acetate (PMA) for 45 min, 37ºC. To verify NETs formation, neutrophils were seeded on cover glasses pretreated with poly-L-lysine in RPMI-1640 medium with or without 100 nM PMA for 45 min, 37ºC. Samples were fixed and later incubated first, with an anti-human myeloperoxidase and then, with Alexa Fluor 488 goat anti-rabbit IgG. Finally, samples were embedded in mounting medium with DAPI and were observed by fluorescence microscopy with a Nikon Eclipse 90i microscope. Cell free DNA (cfDNA) was determined in poor platelet plasma obtained by centrifugation of whole blood (2500 g for 15 min), using the Quant-iT™ Pico Green dsDNA assay (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. To assess thrombin generation associated to NETs, 2.5x105 neutrophils from patients with SLE or from controls were incubated with either buffer or 100 nM PMA during 45 min. Then they were centrifuged at 5000g, 3 min and resuspended in 40-μL of rich platelet rich plasma (PRP) from healthy controls adjusted to 106 platelets/µL obtained from blood samples drawn either in citrate or citrate plus corn trypsin inhibitor (CTI) tubes. CTI is an inhibitor of FXIIa. Calibrated automated thrombogram (CAT) was performed without addition of any trigger. Results: We observed that plasma from patients with SLE had increased free nucleic acids (cfDNA in fluorescence units, controls: 94.90±21.29, SLE patients: 112.4±26.59; P=0.0211). In accordance with this observation, analyses by fluorescence microscopy showed that neutrophils from SLE patients, but not from controls, had NETs even in basal conditions. Moreover, neutrophils from these patients generated more NETs in presence of 100 nM PMA (Figure 1). To evaluate whether the increment of NETs observed in patients with SLE had consequences on the hemostasis of these patients, we tested thrombin generation of neutrophils from either patients with SLE or controls in the presence of platelets from healthy controls. Neutrophils from patients with SLE produced more thrombin than those from healthy controls under basal conditions and after stimulation with 100 nM PMA. These increments were avoided when PRP was collected from blood samples drawn with CTI (Figure 2). Conclusions: Neutrophils from SLE patients without antiphospholipid antibodies and with no history of thrombotic seemed more prone to form NETs than those from healthy controls. NETs might be considered as a key element in the prothrombotic profile of patients with SLE and their analyses by thrombin generation test might be useful to detect risk of occurrence of thrombotic events in these patients and to prevent its occurrence by therapeutic management. This work was supported by grants from FIS-FONDOS FEDER (PI19/00772). EMM holds a predoctoral fellowship from Fundación Española de Trombosis y Hemostasia (FETH-SETH). Disclosures Fernandez-Bello: Stago: Speakers Bureau; Pfizer: Speakers Bureau; SOBI,: Research Funding; Roche: Speakers Bureau; Novartis: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; NovoNordisk: Current Employment, Research Funding, Speakers Bureau. Justo Sanz:Takeda: Current Employment. Alvarez Román:Bayer: Consultancy; Grifols: Research Funding; Pfizer,: Research Funding, Speakers Bureau; SOBI,: Consultancy, Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; NovoNordisk,: Research Funding, Speakers Bureau; Roche: Speakers Bureau; Novartis: Speakers Bureau. García Barcenilla:Novartis: Speakers Bureau; Roche: Speakers Bureau; Pfizer,: Speakers Bureau; NovoNordisk: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Bayer: Speakers Bureau. Canales:Sandoz: Speakers Bureau; Roche: Honoraria; Sandoz: Honoraria; Karyopharm: Honoraria; Roche: Speakers Bureau; Takeda: Speakers Bureau; Roche: Honoraria; Takeda: Speakers Bureau; Novartis: Honoraria; Sandoz: Speakers Bureau; Karyopharm: Honoraria; Roche: Speakers Bureau; Janssen: Honoraria; Janssen: Speakers Bureau; iQone: Honoraria; Sandoz: Honoraria; Gilead: Honoraria; Janssen: Speakers Bureau; Celgene: Honoraria; Janssen: Honoraria; Novartis: Honoraria. Jimenez-Yuste:F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer: Consultancy; F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer, Grifols, Octapharma, CSL Behring, Bayer: Honoraria; Grifols, Novo Nordisk, Takeda, Sobi, Pfizer: Research Funding. Butta:Novartis: Speakers Bureau; NovoNordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; SOBI: Speakers Bureau; Grifols: Research Funding; ROCHE: Research Funding, Speakers Bureau; Pfizer: Speakers Bureau.

scholarly journals Laboratory Characterization of Unclassified Bleeding Disorders By Non-Conventional Tests

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4235-4235
Author(s):  
Paula Acuña ◽  
Elena Monzón Manzano ◽  
Elena G Arias-Salgado ◽  
María Teresa Alvarez Román ◽  
Mónica Martín ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Platelet Function ◽  
Research Funding ◽  
Thrombin Generation ◽  
Bleeding Tendency ◽  
Contact Activation ◽  
Normal Range ◽  
Coagulation Tests ◽  
In Vitro Treatment

Abstract Introduction: Hematologists frequently face a percentage of patients with a mild bleeding tendency due to a haemostatic abnormality that cannot be identified with conventional laboratory techniques. Such patients are termed as having an unclassified bleeding disorder (UBD). A good diagnosis is important in order to prevent bleedings during invasive processes and/or childbirth by choosing the optimal therapeutic treatment. We aimed to investigate hemostatic parameters that may be altered in patients with UBD in order to determine the cause of their bleeding symptoms. In particular, possible defects in the tissue factor (TF)-mediated regulation of coagulation or in the plasmin generation during the fibrinolysis, as well as the possible beneficial effects of treatment with antibodies blockers of TFPI. Methods: This is a single-centre, case-control, non-interventionist, prospective study. During an 8 months-period, 40 patients with bleeding symptoms (evaluated with ISTH-BAT score) were studied. Routine coagulation tests (aPTT and PT) and platelet function testing [aggregometry, PFA-100, flow cytometry and Total Thrombus-formation Analysis System (T-TAS; Zarcos, Japan)] were performed. In 17 patients, no abnormalities were detected in platelet function and/or in coagulation tests; so the following procedures were performed: Thrombin generation test by Calibrated automated thrombography (CAT) in samples of platelet poor plasma with corn trypsin inhibitor (CTI), an inhibitor of contact activation phase, using a low amount of TF (1 pM TF and 4 µM phospholipids) as a trigger to allow the evaluation of the TF-dependent pathway. Plasmin generation (PG) test with a kit from Synapse Research Institute (Maastricht, The Netherlands), using Thrombinoscope software. TFPI activity in plasma, measured with ACTICHROME® TFPI kit (Biomedica Diagnostics, USA). The effects of rFVIIa (Novoseven, NovoNordisk; 90 µg/kg) and of a human Anti-TFPI recombinant Ab (clon mAb2021, Creative Biolabs; 400 ng/ml) were tested in CAT, PG and TFPI activity tests. Results: Those patients with aPTT, PT and a platelet function within normal range were further studied performing thrombin generation, plasmin generation and TFPI activity tests. Table 1 shows the results obtained. Samples from patients 1, 2, 4, 7, 8, 9 and 10 had a diminished generation of thrombin, and in vitro treatment with anti-TFPI and rFVIIa only ameliorated thrombin generation in samples from patients 4, 7, 8 and 9. Plasma from patients 8 and 10 had increased activity of TFPI. Generation of thrombin in samples from patients 3, 5, 6 and 11 was within normal range. Plasmin generation was increased and not modified by in vitro treatment with anti-TFPI and rFVIIa in samples 3 and 11; whereas samples 5 (with normal plasmin generation) and 6 (with no data of plasmin generation due to lack of enough sample) had a high TFPI activity in plasma that was inhibited by anti-TFPI. Normal values in all these parameters evaluated were found in six patients, indicating the involvement of different mechanisms that are still unknown. Conclusions: UBD have a diverse pathological basis for the bleeding. So, a single laboratory test to make a correct diagnosis of this pathology cannot be recommended. In accordance with this fact, a personalized treatment should be applied for each patient. Non-conventional laboratory tests need to be standardized and included for studying possible defects in the regulation of TF and/or plasmin pathways that can be involved in very rare mild bleeding phenotypes. TFPI inhibition might emerge as a good therapy for some of these patients. Failure to detect the bleeding cause in some of these patients, suggests the need to perform further studies in this field. This work was supported by Novo Nordisk Pharma S.A. Table 1- Thrombin and plasmin generation and TFPI activity in samples of patients with UBD. Results out of normal range are shown in red. LT: lagtime; ETP: endogenous thrombin potential; EPP: endogenous plasmin potential; TFPI: Tissue factor pathway inhibitor. Figure 1 Figure 1. Disclosures Alvarez Román: Grifols: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Martín: Novo Nordisk: Speakers Bureau; Pfizer: Speakers Bureau. Jiménez-Yuste: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding. Canales: Eusa Pharma: Consultancy, Honoraria; Sandoz: Honoraria, Speakers Bureau; Sanofi: Consultancy; Karyopharm: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Incyte: Consultancy; Gilead/Kite: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; iQone: Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria. Butta: Novo-Nordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Roche: Speakers Bureau; CSL-Behring: Research Funding.

scholarly journals Some normal value of coagulation tests, proteins concentration tests, iron concentration and copper concentration tests of local house cats in Baghdad city

2012 ◽  
Vol 36 (0A) ◽  
pp. 86-91
Author(s):  
Saleem Amin Hasso
Keyword(s):  
Prothrombin Time ◽  
Protein Concentration ◽  
Copper Concentration ◽  
Iron Concentration ◽  
Fibrinogen Concentration ◽  
Clotting Time ◽  
Total Protein Concentration ◽  
Blood Samples ◽  
Male And Female ◽  
Coagulation Tests

Forty seven blood samples were collected from local house cats 25 male , 22 female , ranging in age from 1-15 year for both sexes for measuring the following parameters coagulation tests (total Platelets count, Clotting time, Prothrombin Time & Fibrinogen concentration ) and proteins concentration tests (Total protein concentration, Albumin , Globulin concentration ) and iron and copper concentration tests .the results as were follow (228.5 –1,537) X 109\L , (2-5)min , (6 - 15) sec , (0.5 -4) g\L , ( 61 - 83 ) g\L , (23.1 -38.2) g\L , (25.9-54.2) g\L , (5.21 – 45.56) μmol/L , (2.29 - 36.52) μmol/L) .the result showed significant differences at level (P<0.05) between the male and female of local house cats in total Platelets count and Prothrombin Time . Other studied parameters showed no significant differences at level (P<0.05) between the male and female in local house cats.

scholarly journals Assessment of Bleeding Phenotype in Hemophilia Α By a Novel Point-of-Care Global Assay

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4662-4662
Author(s):  
Debnath Maji ◽  
Michael A Suster ◽  
Divyaswathi Citla Sridhar ◽  
Maria Alejandra Pereda ◽  
Janet Martin ◽  
...  
Keyword(s):  
Whole Blood ◽  
Research Funding ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Point Of Care ◽  
Blood Samples ◽  
Healthy Group ◽  
Significant Difference ◽  
Bleeding Score ◽  
Bleeding History

Introduction: Patients with Hemophilia A have considerable phenotypic heterogeneity with respect to clinical severity based on their baseline factor levels. As clinical bleeding risk is helpful to individualize factor replacement therapy in hemophilia patients, previous studies have utilized direct and indirect methods of thrombin generation to classify individual bleeding phenotypes, however, with variable results. An easy to use, point-of-care, global assay to assess bleed phenotype, can be a useful tool in the clinical setting to determine intensity of prophylaxis therapy for patients with hemophilia. We have previously introduced a novel, point-of-care (POC), dielectric microsensor, ClotChip, and demonstrated its sensitivity to factor replacement in patients with severe hemophilia A. We aim to further test the ability of ClotChip in assessment of a bleeding phenotype, as described by a bleeding score, in patients with hemophilia A. Methods: After IRB approval, 28 patients with hemophilia A of varying severity and well-characterized bleeding history, were enrolled in this study at the time of trough factor levels. The bleeding history was extracted from patient charts and included number of bleeds (joint and soft-tissue), annual factor usage in terms of units/kg, and number of target joints. These parameters were used to generate a bleeding score (range: 0 - 24), and patients were divided in to 2 categories with scores between 0 - 12 (n=14) and > 12 (n=14). Healthy volunteers (n=17) were accrued as controls. Whole blood samples were obtained by venipuncture into collection tubes containing 3.2% sodium citrate. Samples were then tested with the ClotChip within 2 hours of collection. ClotChip is based on the electrical technique of dielectric spectroscopy (DS) and features a low-cost (material cost < $1), small- sized (26mm × 9mm × 3mm), and disposable microfluidic biochip with miniscule sample volume (< 10 µL). The ClotChip readout was taken as the temporal variation in the real part of blood dielectric permittivity at 1 MHz. Our previous studies have shown that the ClotChip readout is sensitive to the global coagulation process and the time to reach a peak in permittivity (Tpeak) is a sensitive parameter to assess coagulation factor defects. Thrombin generation assay (TGA) using low tissue factor concentration was also performed on blood samples according to the manufacturer's direction. TGA was not available for 4 hemophilia and 2 control samples. Endogenous thrombin potential (ETP) parameter of TGA was used in this study to assess thrombin generation. Data are reported as mean ± standard deviation (SD). Analysis of variance (ANOVA) was used to test for statistical significance between groups with P < 0.05. Spearman's correlation test was used to derive correlation statistics. Results: ClotChip exhibited a mean Tpeak of 2186s ± 1560s for hemophilia patients in the group with higher bleeding scores (i.e. score >12), a mean Tpeak of 931s ± 496s for the group with lower bleeding scores (i.e. score <12) and a mean Tpeak of 441s ± 74s for the healthy group (Figure 1A). A significant difference in Tpeak was found between the group with higher bleeding scores compared to the group with lower bleeding scores (P = 0.002) as well as between higher bleeding scores and the healthy group (P < 0.0001). However, no significant difference in the TGA ETP parameter was detected between the groups with higher bleeding scores (mean ETP: 470 ± 814) and lower bleeding scores (mean ETP: 471 ± 897) (Figure 1B). ETP exhibited a statistical difference between the healthy group (mean ETP: 3462 ± 575) and both hemophilia groups (P < 0.0001). We also carried out studies to investigate the correlative power of the ClotChip Tpeak parameter to the TGA ETP parameter when including additional blood samples that were collected at various times during a hemophilia patient's prophylaxis regimen. The ClotChip Tpeak parameter exhibited strong negative correlation to the TGA ETP parameter (Spearman's rs= -0.73, P < 0.0001). Conclusions: Our studies suggest that a novel dielectric microsensor (ClotChip) could be useful in assessing bleeding phenotype in hemophilia A patients, allowing rapid assessment of hemostasis using a miniscule amount of whole blood (<10 µL) at the POC. Further studies are needed to determine if ClotChip data can be used to individualize prophylactic factor replacement regimens in hemophilia A patients. Disclosures Maji: XaTek, Inc: Patents & Royalties: 9,995,701. Suster:XaTek, Inc: Consultancy, Patents & Royalties: 9,995,701. Mohseni:XaTek, Inc: Consultancy, Patents & Royalties. Ahuja:XaTexk Inc.: Consultancy, Patents & Royalties, Research Funding; Rainbow Children's Foundation: Research Funding; Bayer: Consultancy; Biovertiv Sanofi: Consultancy; Genentech: Consultancy.

scholarly journals Genotype-Phenotype Relationship Among 1200 Unrelated White Patients with Inherited FVII Deficiency: A Three-Center Database Study

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 589-589
Author(s):  
Susan Halimeh ◽  
Gili Kenet ◽  
Piotr Kuta ◽  
Maria Shneyder ◽  
Tido Bajorat ◽  
...  
Keyword(s):  
Blood Group ◽  
Research Funding ◽  
Laboratory Data ◽  
Factor Vii ◽  
Poor Correlation ◽  
Severe Bleeding ◽  
Symptomatic Disease ◽  
Fvii Deficiency ◽  
Work Up ◽  
White Patients

Abstract Background: Congenital factor VII (FVII) deficiency is a rare bleeding disorder caused by mutations in F7 gene with autosomal recessive inheritance. A clinical heterogeneity with poor correlation with FVII:C levels has been described. Aim of this study was to identify genetic defects and to evaluate their relationships with clinical phenotype in a large cohort of 1228 white patients with FVII:C below the age-dependent cut-off %. Methods: Probands with confirmed inherited factor VII deficiency were i) genotyped (Sanger method & multiplex ligation-dependent probe amplification [MLPA]) for F7 mutations including common polymorphic variants and were ii) classified according to clinical bleeding scores (BC). In addition, common thrombophilic variants and blood groups were determined. Results: Probands included asymptomatic subjects (referred for laboratory work up of recurrent prolonged prothrombin time; n=415; mean age 30 + 19 yrs.; female 52%) and patients who presented with mild, moderate or severe bleeding episodes (n=805; mean age 34 + 18 yrs.; female 70%). Bleeding symptoms included epistaxis, gum bleeding, GI bleeding, hematuria, postoperative and gynecologic hemorrhage. Median FVII activity (entire cohort) measured with clotting-based assays (ACL TOP 750 and/or BCS-XP) was 49% (range 5-78%) and the median ISTH BS recorded within a period of two years prior to work-up was 1(0-17). The latter was significantly higher in women compared with males (2 versus 1; p &lt; 0.001). The corresponding PBAC-Score prior to hormonal treatment was 163 (25-1200). Blood group 0 was present in 40.6% of cases. Known and novel mutations in the F7 gene, including coding regions, exon/intron boundaries and the promoter region, are found in 534 patients (44%) and common polymorphisms were detected in 666 subjects (95%). Logistic regression analysis adjusted for clinical and laboratory data (blood group, FVII activity, presence of F7 gene mutations and /or polymorphisms, thrombophilia status and further factor deficiencies) revealed that older age (increase per year) at referral (odds/95% CI: 1.01/1.007-1.025) and female gender (odds/95% CI: 3.25/2.35-4.50) in part explain differences in clinical bleeding phenotype associated with FVII deficiency. Conclusion: Overall there is poor correlation between the FVII level and the bleeding phenotype. Older patients and females are more likely to have symptomatic disease as a result of gynecological or muco-cutaneous bleeding. Disclosures Kenet: Alnylam: Consultancy, Research Funding; Bayer: Consultancy, Research Funding; Opko Biologics: Research Funding; Pfizer: Consultancy, Research Funding; Shire: Research Funding; Novo Nordisk: Consultancy; Roche: Consultancy; Takeda: Consultancy.

scholarly journals Sufficient Thrombin Generation Despite 95% Hemodilution: An In Vitro Experimental Study

2020 ◽  
Vol 9 (12) ◽  
pp. 3805
Author(s):  
Johannes Gratz ◽  
Christoph J. Schlimp ◽  
Markus Honickel ◽  
Nadine Hochhausen ◽  
Herbert Schöchl ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Prothrombin Complex Concentrate ◽  
Oral Anticoagulants ◽  
Coagulation Factor ◽  
Severe Bleeding ◽  
Crystalloid Solution ◽  
Coagulation Tests ◽  
Coagulation Factor Concentrates ◽  
Dilution Level

Guidelines for the treatment of severe bleeding comprise viscoelastic-test-guided use of coagulation factor concentrates as part of their recommendations. The aim of this study is to investigate the effects of substituting fibrinogen, prothrombin complex concentrate, and a combination of both on conventional coagulation tests, viscoelastic test results, and thrombin generation. Blood was drawn from seven healthy volunteers to obtain platelet-free plasma, which later was diluted by replacing 40%, 60%, 80%, 90%, 95%, and 99% with a crystalloid solution. The diluted samples were spiked with fibrinogen concentrate, prothrombin complex concentrate, a combination of both, or a corresponding amount of crystalloid solution. Up to a dilution level of 95%, viscoelastically determined clotting time was significantly shorter in the group substituted with fibrinogen only in comparison with the additional use of prothrombin complex concentrate. Clot firmness and endogenous thrombin potential remained at relatively stable values up to a dilution level of 95% with the substitution of fibrinogen but not prothrombin complex concentrate. Substitution of prothrombin complex concentrate led to an excessive overshoot of thrombin generation. The results of our study question currently propagated treatment algorithms for bleeding patients that include the use of prothrombin complex concentrate for patients without former intake of oral anticoagulants. Even in severely bleeding patients, thrombin generation might be sufficient to achieve adequate hemostasis.

scholarly journals The Sonoclot: A New Thrombokinetic Coagulation Instrument

1977 ◽  
Author(s):  
R.D. Hamstra ◽  
G.E. Ens ◽  
S. Simons
Keyword(s):  
Whole Blood ◽  
Blood Clotting ◽  
Formation Rate ◽  
Severe Bleeding ◽  
Axial Vibration ◽  
Clotting Time ◽  
Activated Clotting Time ◽  
Coagulation Tests ◽  
Disposable Plastic ◽  
Normal Adults

A new coagulation instrument (Sonoclot) has been evaluated in the laboratory, at the bedside, and in surgery. It was compared to the prothrombin time, activated partial thromboplastin time, activated clotting time, Lee-White clotting time, fibrinogen and thrombelastograph. Most coagulation tests are reported as time to a clot end point while the Sonoclot records a continuous thrombokinetic pattern by measuring the energy required to maintain axial vibration of a hollow plastic probe in 0.4 ml of whole blood while it clots. Disposable plastic probes and non-siliconized glass cuvettes are rapidly changed after testing. Results are a line pattern which has been analysed for Clot Onset (C0) and Clot Formation Rate (CFR). Native whole blood from normal adults (54 studied) has a C0 of 2-4.5 minutes and CFR of 4-8 chart units/minute. The only similar clotting instrument is the thrombelastograph (TEG). Sonoclot and TEG patterns correlate, the Sonoclot sensing change before the TEG. Whole blood clotting measured with the Sonoclot is a new method which distinguishes hypercoagulation, various levels of heparin, and severe bleeding problems from normal. The device is durable, stable, easy to operate, and portable, providing a permanent record in a few minutes.

scholarly journals Distinct Localization of Coagulation Factor VIII, Von Willebrand Factor and Factor VIIIa-Mimetic Bispecific Antibody Contributing to Thrombus Formation Under Whole Blood Flow Conditions

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1483-1483
Author(s):  
Yasuaki Shida ◽  
Keiji Nogami ◽  
Hiroaki Minami ◽  
Hiroaki Yaoi ◽  
Tomoko Matsumoto ◽  
...  
Keyword(s):  
Shear Flow ◽  
Research Funding ◽  
Hemophilia A ◽  
Thrombus Formation ◽  
Flow Chamber ◽  
High Shear ◽  
Flow Conditions ◽  
Von Willebrand ◽  
Low Shear

Abstract Background Factor VIII (FVIII) is an essential factor for coagulation system in the intrinsic pathway. Due to the short survival of FVIII in the plasma circulation, it requires von Willebrand factor (VWF) as a carrier protein to maintain the optimal level for hemostasis. VWF also plays an important role in primary hemostasis by bridging platelets to exposed subendothelial collagens, especially under high shear flow environment. Since VWF carries FVIII, it is conceivable that VWF takes FVIII to the sites of vascular injury. However, the role of FVIII at the local sites under flow conditions is not fully understood despite of the fact that increased level of FVIII is associated with the risk of venous thrombosis and the deficiency of FVIII is the pathology of the bleeding disorder, hemophilia A. The treatment of hemophilia A largely depends on the infusion of FVIII concentrates, which is often complicated by the development of the inhibitor. Recently, bispecific antibody（ACE910）that mimics the role of FVIIIa by recognizing FIXa and FX has been developed and is currently under clinical trial. This antibody theoretically works regardless of the presence of devastating inhibitors against FVIII. Furthermore, it could also improve the clinical outcome of the other bleeding disorders, such as von Willebrand disease (VWD). Aim To analyze the role of FVIII and VWF, and impact of ACE910 at the sites of vascular injury under various shear conditions, we have developed the flow-mediated thrombosis model using flow chamber system. Method Whole blood obtained from healthy donors, hemophilia A and VWD patients were perfused into the collagen coated flow chamber under high (2,500s-1) or low shear (50s-1) flow conditions with/without FVIII concentrate, FVIII/VWF concentrate and ACE910. Formed thrombus was fixed and immunostaining was performed with phalloidin (Platelet), anti-FVIII antibody (FVIII) and anti-thrombin antibody (Thrombin). For the detection of ACE910, anti-human IgG or anti-ACE antibody (rAQ8 or rAJ540) were used. Size of thrombi and distribution of platelet, FVIII, thrombin and ACE910 were analyzed. Result 1) Under high shear flow, thrombus formation of VWD blood was significantly impaired while blood from Hemophilia A demonstrated nearly normal thrombus formation. Addition of FVIII/VWF but not FVIII concentrate to the blood of these patients rescued the impaired thrombus formation. ACE910 enhanced the thrombus formation of blood from both VWD and hemophilia A. Under low shear flow, blood from both hemophilia A and VWD demonstrated decreased thrombus formation. FVIII, FVIII/VWF concentrates and ACE910 improved the size of thrombus. 2) Localization of FVIII was evaluated with thrombin as a marker for the activation of coagulation. Platelets and thrombin demonstrated complete co-localization and intensity of thrombin staining was associated with thrombus size. VWF localized mainly outer layer of thrombus and FVIII localized in and around thrombus. At high shear condition, FVIII and VWF mostly existed with platelets. By contrast, FVIII and VWF demonstrated less co-localization with platelets under low shear condition. ACE910 demonstrated similar tendency to FVIII localization although ACE910 did not appear around thrombus. Conclusion We have developed the flow chamber system to evaluate the extent of thrombogenesis under various shear environment. VWF showed dominant role under high shear conditions while FVIII plays a key role under low shear conditions. FVIII, VWF and ACE910 demonstrated distinct localization. Interestingly, the distribution of FVIII was broader than VWF and platelet. FVIII localized to platelets presumably prior to its activation and contributed for the subsequent thrombin generation at local sites. Finally, ACE910 demonstrated consistent enhancement of thrombus formation of blood from both hemophilia A and VWD and, therefore, is prompted for the treatment of these bleeding disorders. Disclosures Shida: Chugai Pharmaceutical Co., Ltd.: Research Funding. Nogami:Chugai Pharmaceutical Co., Ltd.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Minami:Chugai Pharmaceutical Co., Ltd.: Research Funding. Yaoi:Chugai Pharmaceutical Co., Ltd.: Research Funding. Matsumoto:Chugai Pharmaceutical Co., Ltd.: Research Funding. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Hattori:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Shima:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Evaluation of the REG1 Anticoagulant System Using Thrombelastography and Thrombin Generation Assay.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4010-4010
Author(s):  
Kenichi A. Tanaka ◽  
Fania Szlam ◽  
Christopher P. Rusconi ◽  
Jerrold H. Levy
Keyword(s):  
Whole Blood ◽  
Thrombin Generation ◽  
Thrombus Formation ◽  
Point Of Care ◽  
Phase 1 ◽  
Lag Time ◽  
Blood Draw ◽  
Blood Samples ◽  
Thrombin Generation Assay ◽  
Anticoagulant System

Abstract Background: The REG1 system (Regado Biosciences, Inc. Durham, NC) is a novel anticoagulant system which comprises RB006 (drug) and RB007 (antidote). The aptamer, RB006 selectively binds to FIXa and blocks factor Xa generation. RB007 is a complementary ligand that selectively binds to RB006, and reverses its anticoagulant effect (1). Phase 1 testing demonstrated a clear pharmacodynamic dose response to RB006 in plasma coagulation assays (1), but further work is needed to understand the pharmacodynamic response to the REG1 system in whole blood assays. Therefore, we evaluated the effects of RB006 and RB007 alone and in combination using activated partial thromboplastin time (APTT), viscoelastic (TEG®, Haemoscope, IL) and thrombin generation assay (Thrombinoscope™, Synapse BV). Methods: After IRB approval, blood samples were collected from 4 consented healthy volunteers into 3.2% citrate tubes. For APTT and TEG testing blood was placed in thirteen 2 ml Eppendorf tubes. Tube one had 20 μl of saline added and served as a control. The remaining 12 tubes were divided into 3 groups. Group1: RB006 (final concentrations 3, 6, 12, 18 and 24 μg/ml); Group 2: RB007 (final concentrations 6, 12, 24, and 48 μg/ml) and Group 3: combination of both agents at a weight:weight ratio 2 to 1 for RB007:RB006 (final concentrations as above). All testing was performed within 3-hour of blood draw. APTT was done using Hemochron Jr® (ITC, NJ) instrument in recalcified whole blood. TEG was peformed in recalcified whole blood, 360 μl activated with 2 nM thrombin. For Thrombinoscope, whole blood was centrifuged at 2000 x g for 15 min to obtain platelet poor plasma (PPP). PPP samples were prepared to contain the same concentrations of RB006, RB007, and combination of both agents as described in whole blood samples. Thrombin generation analyses were performed using microplate format using diluted Actin (Dade Behring, Marburg, Germany) as a trigger (2). Results: RB006 dose dependently increased APTT (Table 1). Increasing concentrations of RB006 progressively prolonged onset, and decreased the rate of thrombus formation on TEG (Figure1A). On Thrombinoscope, RB006 dose dependently delayed lag time and decreased peak thrombin generation (Table 1, Figure1B). All the parameters of aPTT, TEG and thrombin generation returned back to control values when combination of aptamer-anti-aptamer was tested. RB007 alone had no effects on any of the tests performed. Conclusion: Along with conventional point of care APTT testing TEG and Thrombinoscope methodologies can be very useful in monitoring the anticoagulation effects of aptamer, RB006, and its reversal with anti-aptamer, RB007. Effects of Aptamer, RB006, on aPTT and Thrombinoscope lag time/peak thrombin RB006μg/ml APTT sec Lag time min Peak thrombin nM Data shown as mean ± SD 0 (control) 51.3 ± 1.0 10.8 ± 0.3 244 ± 16.2 3 95.3 ± 3.0 14.2 ± 0.9 173 ± 22.9 6 152 ± 10.0 18.8 ± 0.5 145 ± 23.0 12 183 ± 4.3 24.1 ± 1.1 105 ± 11.0 18 238 ± 15.3 26.3 ± 2.3 92.3 ± 7.3 24 257 ± 11.3 31.3 ± 3.8 88.2 ± 8.3 Figure 1 Figure 1.

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