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 A Novel Point-of-Care Whole Blood Coagulation Assay to Monitor Emicizumab Therapy in Patients with Hemophilia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2475-2475 ◽  
Author(s):  
Michael A Suster ◽  
Debnath Maji ◽  
Lalitha V. Nayak ◽  
Christina Jenkins ◽  
Susan Hunter ◽  
...  
Keyword(s):  
Whole Blood ◽  
Research Funding ◽  
Hemophilia A ◽  
Ex Vivo ◽  
Point Of Care ◽  
Blood Samples ◽  
University Patents ◽  
Whole Blood Samples ◽  
Western Reserve

Abstract Introduction: Emicizumab is a humanized, bispecific antibody against activated Factor IX (FIX/IXa) and FX/Xa that mimics the cofactor function of activated FVIII (FVIIIa) by spatially relocating FIXa and FX to the appropriate position in the tenase complex. Though the bleeding rate in patients on emicizumab is remarkably reduced, hemostatic monitoring is important in the event of breakthrough bleeding, development of anti-drug antibodies, and for surgery. An APTT is markedly shortened in the presence of small amounts of emicizumab, and, hence not useful. The thrombin generation assay (TGA), thromboelastometry, and clot waveform analysis measure the hemostatic effect of emicizumab, both in vivo and ex vivo. However, these assays are time-consuming, need expert interpretation, and not widely available. We have developed a novel, point-of-care, whole blood assay based on a dielectric microsensor (ClotChip) that can obtain global blood coagulation assessment in a miniaturized, portable measurement platform. The aim of this study was to assess the sensitivity of ClotChip to detect the addition of variable concentrations of emicizumab ex vivo into hemophilic whole blood and the feasibility of monitoring emicizumab therapy in vivo using ClotChip . Methods: After IRB approval, patients accrued for the study were adults with severe hemophilia A with inhibitors starting emicizumab therapy (n=2), and a child with severe hemophilia A without inhibitors (n=1). Blood samples were obtained by venipuncture into collection tubes containing 3.2% sodium citrate anticoagulant. Samples were collected both prior to (n=2), and at time points of 30 min (n=1), 1 hour (n=1), 1 week 1 (n=1), and 8 weeks (n=1) after initiation of emicizumab therapy. Whole blood samples from hemophilia patients not on emicizumab were spiked with different concentrations of emicizumab, with and without the addition of replacement therapy. Whole blood samples were then tested with the ClotChip. Coagulation was induced with CaCl2 addition. ClotChip is based on the electrical technique of dielectric spectroscopy (DS) integrated into a low-cost (material cost < $1), small- sized (26mm × 9mm × 3mm), and disposable microfluidic biochip with miniscule sample volume (< 10 µL). ClotChip curves were calculated as blood permittivity at 1MHz, and the time to reach a peak in permittivity (TpeakFig 1A) was taken as an indication of coagulation time. TGA using low tissue factor concentration was also performed on blood samples according to the manufacturer's direction. Results: We observed a decrease in the ClotChip Tpeak parameter for post-therapy samples (30 min and 1 hr.) compared to baseline (pre-therapy) for hemophilia patients with inhibitors on emicizumab therapy (Fig 1B). A time-dependent decrease was observed in ClotChipTpeak after emicizumab administration with week 1 and 8 samples showing normal values. Ex vivo spiking with emicizumab in blood from patients with hemophilia with and without inhibitors showed a concentration dependent decrease in ClotChip Tpeak parameter (Fig 1C). Addition of rFVIII or rFIX in emicizumab-spiked blood from the patient without inhibitors further decreased ClotChipTpeak in a concentration dependent manner (Fig 1D). Similarly, addition of rFVIII to the emicizumab spiked blood from the inhibitor patient further decreased ClotChip Tpeak compared to emicizumab alone (Fig 1E). ClotChip Tpeak exhibited strong negative correlation with ETP (rs = 0.81, Fig 1F) and peak thrombin (rs = -0.82) from TGA assays. Conclusions: Our studies demonstrate the feasibility of monitoring emicizumab therapy in patients with hemophilia with and without inhibitors, using a novel, microfluidic, dielectric sensor (ClotChip), allowing whole blood assessment of hemostasis in a single disposable sensor. ClotChip has potential to fulfill an unmet clinical need to assess global coagulation potential in hemophilia patients on emicizumab therapy, especially when additional hemostatic therapy is needed for treatment of breakthrough bleeds. Additional spiking studies to assess the addition of bypassing agents (rFVII or FEIBA) to blood from emicizumab-treated patients are underway. Disclosures Suster: Case Western Reserve University: Patents & Royalties: licensed to XaTek, Inc.; XaTek, Inc.: Consultancy. Maji:Case Western Reserve University: Patents & Royalties: Licensed to XaTek, Inc. . Schmaier:Temple University: Patents & Royalties; Shire: Consultancy, Honoraria, Research Funding; Enzyme Research Laboratories: Honoraria; Cleveland Clinic Foundation: Research Funding; Alnylam: Research Funding; Biomotiv: Consultancy. Mohseni:Case Western Reserve University: Patents & Royalties: Licensed to XaTek, Inc.; XaTek, Inc.: Consultancy. Ahuja:Shire: Honoraria, Speakers Bureau; Bayer: Honoraria; Bioverativ: Honoraria, Speakers Bureau.

Thromboelastography in Children with Coagulation Factor Deficiencies.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2139-2139 ◽  
Author(s):  
Meera B. Chitlur ◽  
Indira Warrier ◽  
Madhvi Rajpurkar ◽  
Wendy Hollon ◽  
Lolita Llanto ◽  
...  
Keyword(s):  
Whole Blood ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Response To Treatment ◽  
Factor Vii ◽  
P Value ◽  
Factor V ◽  
Mean Values ◽  
Significant Difference

Abstract The thromboelastograph produces a continuous profile of the rheological changes that occur during the process of coagulation using whole blood. This information can be transformed into a dynamic velocity profile of the changes in blood elasticity occurring during clotting. We used the TEG® hemostasis analyzer in patients with hemophilia A or B with and without inhibitors and other coagulation factor deficiencies (OFD), to study the thromboelastographic profiles in these patients. Materials and Methods: 62 children (6 months-19 years old) were enrolled according to IRB regulations. 29 children had severe hemophilia A (SHA), 4 moderate hemophilia A or B (Mod.H), 2 severe factor VII deficiency, 1 combined factor V and VIII deficiency, 1 VWD (type II B), 1 severe factor V deficiency, 1 Severe PAI deficiency, 19 normal controls (NC), and 4 SHA with inhibitors (SHA+I). All patients were studied 72 hours after the last dose of factor. Citrated whole blood was activated using recombinant human tissue factor (Innovin, Dade Behring Inc®) and recalcified using 0.2M CaCl2. In patients with central lines with heparin, a heparinase cup was used. The TEG® was run for ≥ 90 min. CBC with differential was obtained on all subjects. Results: There was no significant difference in the CBC parameters among patients. Analysis of the TEG data revealed the following: Table 1 TEG Parameters (mean values) SHA (n=29) Mod.H (n=4) SHA+I (n=4) OFD (n=6) Control(n=19) MTG:Max rate of thrombin generation; TMG: Time to MTG; R: Reaction Time; K: Time to reach an amplitude of 20mm; MA: Max. Amplitude MTG(mm*100/sec) 8.7 9.6 1.3 9 17 TMG(min) 27.5 16.6 62.7 17.5 8.9 R(min) 22 14 56 15 7 K(min) 7 4 41 4 2 Max.Amplitude, MA (mm) 59 56 12 58 62 The rate of thrombin generation as visualized by plotting the 1st derivative of the TEG course, in patients with SHA without inhibitors, showed that they could be divided into 2 groups based on MTG (</>9). When analysed the 2 groups showed the following characteristics (5 representative curves from each group are shown): Figure Figure Table 2 TEG Parameters (Mean values) MTG < 9 (n=16) MTG > 9 (n=13) p value TMA: Time to MA; MTG(mm*100/sec) 5.5 12.6 <0.001 TMG (min) 33 20 0.009 R(min) 26 16 0.004 K(min) 9 3.4 0.03 MA(mm) 56.1 62.3 0.01 TMA(min) 60 38 0.006 13/29 children with SHA had target joints and 69%of patients with target joints had a MTG<9. Conclusions: SHA patients have variable bleeding tendencies as seen by the variation in MTG. A lower MTG is associated with a higher incidence of target joints. This may provide a clue as to which patients may have the greatest benefit from primary prophylaxis. Patients with OFD have a TEG® profile similar to Mod.H patients. SHA+I have poor thrombin generation as seen by a significantly longer TMG and R time (p <0.05), compared to all subjects. The TEG may provide valuable clues to the severity of bleeding tendencies in patients with factor deficiencies. In additional observations (not shown), it appears that the TEG may be used to monitor the response to treatment with factor concentrates and tailor treatment with rFVIIa.

Hemophilia a Clots Generated with Recombinant Factor VIIa Differed in Structure and Composition from Those Formed with Factor VIII

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3798-3798
Author(s):  
Lilley Leong ◽  
Irina N. Chernysh ◽  
Yifan Xu ◽  
Cornell Mallari ◽  
Billy Wong ◽  
...  
Keyword(s):  
Factor Viii ◽  
Whole Blood ◽  
Neutralizing Antibodies ◽  
Research Funding ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Clot Formation ◽  
Factor Vii ◽  
Wild Type ◽  
Independent Mechanism

Abstract Patients with severe factor VIII (FVIII) deficiency (hemophilia A [HemA]) develop neutralizing antibodies (inhibitors) against FVIII in up to ~30% of cases. For HemA patients with inhibitors, activated recombinant factor VII (rFVIIa) is a treatment option. High levels of rFVIIa are required for treating HemA patients with inhibitors to induce direct activation of factor X on the surface of activated platelets via a tissue factor (TF)-independent mechanism (Hoffman M, Monroe DM. Thromb Res. 2010;125(suppl 1):S16-S18). To assess how rFVIIa-mediated clot formation in HemA patients with inhibitors may differ from unaffected individuals, we compared the effect of rFVIIa on HemA versus control (or HemA supplemented with 100% FVIII) clot formation in human and/or mouse systems. By TF-induced thrombin generation assay, increasing rFVIIa from 5 nM to 100 nM did not appreciably alter the kinetics or extent of thrombin generation compared with the same human HemA plasma containing 100% FVIII. Confocal microscopy of human HemA plasma clots generated with 75 nM rFVIIa and TF showed few branching fibrin fibers and an open fibrin meshwork. In contrast, TF-induced coagulation of the same HemA plasma containing 100% FVIII formed fibrin clots with numerous branches, interconnecting to form a dense meshwork. To confirm that these findings reflect rFVIIa-mediated clot formation in vivo, we assessed the intrinsic coagulation of mouse HemA whole blood collected without anticoagulant and spiked with rFVIIa. Intrinsic coagulation with rFVIIa was assessed by T2 magnetic resonance (T2MR), a technique capable of monitoring the separation of whole blood into serum, loose-clot, and tight-clot compartments during coagulation (Skewis et al. Clin Chem. 2014;60:1174-1182; Cines et al. Blood. 2014;123:1596-1603). By T2MR, rFVIIa induced the separation of HemA whole blood into the serum and clot compartments, indicating that the reduced fibrin generation with rFVIIa did not interfere with whole blood coagulation. Furthermore, saphenous vein puncture of HemA mice treated with rFVIIa showed a dose-dependent decrease in clot times. Scanning electron microscopy of the clots extracted from these HemA mice indicated markedly different composition than clots extracted from wild-type mice. In wild-type clots, fibrin and polyhedral erythrocytes formed a large proportion of the total structures. In contrast, clots from rFVIIa-treated HemA mice consisted primarily of platelets and erythrocytes with forms intermediate between discoid and polyhedral but, surprisingly, low fibrin content. Taken together, these data suggest that rFVIIa-mediated clot formation may require greater activated platelet involvement, which would be consistent with the TF-independent mechanism of action proposed for rFVIIa in HemA. Finally, the compositional difference between clots from wild-type versus HemA mice dosed with rFVIIa suggest that evaluating HemA therapies for their ability to form more physiologic clots could be an approach to improve treatment options for patients with HemA. Disclosures Leong: Bayer: Employment. Xu:Bayer: Employment. Mallari:Bayer: Employment. Wong:Bayer: Employment. Sim:Bayer: Employment. Cuker:Stago: Consultancy; Genzyme: Consultancy; Amgen: Consultancy; Biogen-Idec: Consultancy, Research Funding; T2 Biosystems: Research Funding. Marturano:T2 Biosystems: Employment. Lowery:T2 Biosystems: Employment. Kauser:Bayer: Employment. Weisel:Bayer: Research Funding.

Hypercoagulability in Sickle Cell Disease: The Importance of the Cellular Component of Blood

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4060-4060 ◽  
Author(s):  
Matthew F Whelihan ◽  
Ming Y. Lim ◽  
Bethany L Walton ◽  
Alisa S. Wolberg ◽  
Jianwen Cai ◽  
...  
Keyword(s):  
Whole Blood ◽  
Maximum Rate ◽  
Thrombin Generation ◽  
Maximum Level ◽  
Cell Disease ◽  
Contact Activation ◽  
Blood Samples ◽  
Significant Difference ◽  
Cellular Components ◽  
Clot Time

Abstract Sickle cell disease (SCD) is considered to be a hypercoagulable state with chronic activation of coagulation and an increased incidence of thrombotic events. However, there is no consensus on whether global assays of thrombin generation in platelet poor (PPP) or platelet rich (PRP) plasma display an increased thrombin generation potential in SCD (reviewed in Lim et al. Curr. Opin. Hematol. 2013). Based on our recent observation that RBC contribute to thrombin generation in whole blood (Whelihan et al. Blood 2012), we hypothesized that the cellular components in blood (notably RBCs) contribute to enhanced thrombin generation in SCD. 25 SCD patients in a non-crisis, “steady state” and 25 healthy race–matched controls were recruited for study. Whole blood thrombin generation, thromboelastography (TEG) and plasma-based thrombin generation assays (TGA) were performed on contiguous blood samples from each individual. Complete blood counts, as well as quantification of phosphatidylserine (PS) exposure by RBCs (assayed by Annexin V binding in flow cytometry) were also performed. Whole blood thrombin generation was monitored by serial α-thrombin-antithrombin (TAT) complex formation following activation by both the extrinsic pathway (5 pM recombinant tissue factor (TF)) and intrinsic (native contact activation) pathways. Results. With extrinsic activation, controls clotted on average at 3.9±0.5 min (mean±SD), generated TAT at a rate of 38.8±22.2 nM/min and reached a maximum level of 261±58nM. In patients with SCD, clot time (4.4±0.8 min, ƿ=0.04) and the maximum rate of TAT generation (41.5±19.4 nM/min, ƿ=0.65) were similar to that observed in controls while the maximum level (369±123 nM) of TAT generated was significantly higher (ƿ=0.0026). With contact activation, there was no significant difference in clot time (7.2±1.2 min vs 6.4±1.0 min, ƿ=0.07) or the maximum rate of TAT generation (52.9.5±26.9 nM/min vs 42.3 ± 12.4 nM/min, ƿ=0.57) for the SCD and control cohorts, respectively. However, similar to what was observed with extrinsic activation, SCD patients generated a significantly higher (ƿ=0.024) maximum level of TAT (352±116 nM) than controls (276±21 nM). Interestingly, the SCD cohort showed a strong positive correlation (ƿ=<0.001) between the maximum levels of TAT generated with either stimulus (extrinsic vs intrinsic). We also examined extrinsic- and intrinsic-initiated clot formation using TEG. Re-calcification of citrated blood from SCD in the presence of TF and Corn Trypsin Inhibitor (CTI) displayed similar R (8.1±0.3 min vs 8.4±0.6 min, ƿ=0.7) and MA (68±2 mm vs 65±2 mm, ƿ=0.16) values to those exhibited by controls, respectively. The -angle however, was significantly (ƿ=0.035) higher in the SCD cohort (61±3°) compared to controls (53±3°). Contact activated blood also displayed no significant difference in R time between the two groups (14.5±3 min for SCD vs 16.5±4 min for controls). On the other hand, the MA and -angle were significantly increased (ƿ=0.04 and ƿ=0.012) in SCD (62±2 mm and 41±3°) compared to the controls (51±3 mm and 26±5°), suggesting a significant increase in the overall rate and extent of clot formation as a result of contact activation. To examine thrombin generation in the absence of cellular components, PPP was activated using an identical 5pM TF stimulus. In contrast to whole blood, TGA displayed no significant differences in the peak thrombin or maximum rate of thrombin generation between the two groups. Lag time was marginally longer and time to peak thrombin generation shorter in SCD (ƿ=0.036 and ƿ=0.03, respectively). Surprisingly, a weak negative correlation (ƿ=0.15) between RBC PS expression and total TAT was present in SCD patient samples. Conclusions: While plasma-based assays exhibit no major differences in thrombin generation potential between SCD and controls, corresponding whole blood samples showed a significant increase in overall thrombin generation and clotting potential, regardless of the initiating stimulus (extrinsic or intrinsic). Interestingly, there was no significant correlation between absolute cell counts (i.e. RBCs, Retics, Neuts, PLTs) or parameters and TAT levels. Collectively, these data make a strong case for cellular involvement in the hypercoagulability observed in SCD, but a direct role for RBC PS expression in net thrombin generation is not apparent. Disclosures No relevant conflicts of interest to declare.

A Native Whole Blood Thrombin Generation Assay Allows Discrimination of Whole Blood Samples with FVIII Levels below 1%

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4215-4215
Author(s):  
Christina K Baumgartner ◽  
Jonathan C Roberts ◽  
Paula M Jacobi ◽  
Sandra L Haberichter ◽  
Qizhen Shi ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Blood Samples ◽  
Time Points ◽  
Fviii Activity ◽  
Post Infusion ◽  
Whole Blood Samples ◽  
Dose Dependent

Abstract Monitoring the correction of abnormal bleeding tendencies during the treatment of patients with hemostatic disorders is essential to evaluate success of therapy. While single clotting factor assays provide valuable information, global coagulation assays are desirable to better understand the overall hemostatic condition of patients. In Hemophilia A, severity of the clotting defect is traditionally evaluated by determining FVIII activity using chromogenic or clotting assays. Evaluation of thrombin generation in plasma samples for the assessment of bleeding tendencies in hemophilic patients has been suggested. Discriminating between samples with FVIII levels below 1%, however, has been challenging using FVIII activity and thrombin generation assays. We previously reported a native whole blood thrombin generation assay (nWB-TGA) that uses recalcification of whole blood samples without the addition of tissue factor to initiate clotting. We have shown that this assay is sensitive to varying levels of FVIII in vitroand to platelet targeted FVIII gene therapy in a murine model of Hemophilia A. The objective of the present study was to determine if the nWB-TGA can be used to monitor Hemophilia A patients during FVIII therapy and if this assay allows discrimination of whole blood samples with FVIII levels below 1%. Using the nWB-TGA we evaluated thrombin generation in a severe hemophilia A patient carrying an intron 22 inversion. Numerous data points were obtained from 15 different FVIII infusions, each targeting a FVIII level of 50%. Samples collected at least 72 hours (hrs) post infusion (>6 half-lives, calculated FVIII levels <1%) significantly differed from healthy control samples in all thrombin generation parameters. Compared with healthy controls (6.9 ± 0.6 min; mean ± SEM) the hemophilic patient had a lag time (LT) of 24.8 ± 3.4 min. Peak time in healthy controls and the patient was 10.1 ± 1 min and 35 ± 5 min, peak thrombin was 528 ± 78 nM and 124 ± 20 nM, endogenous thrombin potential (ETP) was 1949 ± 117 nM and 1201 ± 50 nM, and thrombin generation rate was 196 ± 58 nM/min and 21 ± 6 nM/min, respectively. While previous studies on thrombin generation in plasma samples mainly reported on differences in peak thrombin and ETP, spiking of hemophilic blood with increasing concentration of recombinant FVIII in vitro revealed that the LT was FVIII dose dependent in our assay. When hemophilic blood was reconstituted with FVIII to a 2%, 5% and 50% level, the LT was 22.5 ± 1.6 min, 16.1 ± 1.7 min and 8.8 ± 0.6 min, respectively. All other thrombin generation parameters were FVIII dose dependent as well. A FVIII dependent LT was also apparent in vivo, when we monitored the patient after FVIII infusions. LT was 6.4 ± 0.2 min at 15 min, 8.5 ± 0.4 min at 24 hrs, and 13.8 ± 0.5 min at 48 hrs post FVIII treatment. We identified the lack of tissue factor as being key to a FVIII dose dependent LT. At all post infusion time points the LT was approximately 5 min when tissue factor was added to the assay. To our surprise, looking at specific time points equal to or greater than 72 hrs post infusion enabled us to discriminate these samples based on the LT (72 hrs: LT= 13.0 ± 0.6 min, 84 hrs: LT= 19.5 ± 0.8 min, 96 hrs: 36.0 ± 4.4 min). The ETP, commonly used as a variable parameter in previous thrombin generation reports, however, was not different among 72, 84 and 96 hrs post FVIII infusion samples. Strikingly, FVIII activity determined by chromogenic and one stage clotting assay was below detection limit (1% FVIII:C) in samples obtained 72 hrs post infusion or later. Thus, the patient in our study displayed considerable thrombin generation determined by the nWB-TGA at post FVIII infusion time points when FVIII levels were considered undetectable with currently available technology. Our data suggest that the different LT observed in 72, 84 and 94 hrs post infusion samples is possibly related to differences in residual FVIII levels below 1%. In conclusion, the nWB-TGA provides a useful tool to monitor efficacy of FVIII replacement therapy and might assist in tailoring individual FVIII treatment regimens. This close to physiological whole blood assay allows distinguishing blood samples with FVIII levels below 1% in vivo, and might help to explain the heterogeneity in bleeding phenotypes observed in severe hemophilia A patients. This assay may also be useful in assessing therapeutic benefit of “long acting” FVIII or FIX products. Disclosures No relevant conflicts of interest to declare.

scholarly journals Monitoring Thrombin Generation by Electrochemistry: Development of an Amperometric Biosensor Screening Test for Plasma and Whole Blood

2009 ◽  
Vol 55 (3) ◽  
pp. 505-512 ◽  
Author(s):  
Charles Thuerlemann ◽  
André Haeberli ◽  
Lorenzo Alberio
Keyword(s):  
Whole Blood ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Point Of Care ◽  
Electrical Signal ◽  
Measuring System ◽  
Screening Tests ◽  
Blood Samples ◽  
Biosensor System ◽  
Whole Blood Samples

Abstract Background: Complete investigation of thrombophilic or hemorrhagic clinical presentations is a time-, apparatus-, and cost-intensive process. Sensitive screening tests for characterizing the overall function of the hemostatic system, or defined parts of it, would be very useful. For this purpose, we are developing an electrochemical biosensor system that allows measurement of thrombin generation in whole blood as well as in plasma. Methods: The measuring system consists of a single-use electrochemical sensor in the shape of a strip and a measuring unit connected to a personal computer, recording the electrical signal. Blood is added to a specific reagent mixture immobilized in dry form on the strip, including a coagulation activator (e.g., tissue factor or silica) and an electrogenic substrate specific to thrombin. Results: Increasing thrombin concentrations gave standard curves with progressively increasing maximal current and decreasing time to reach the peak. Because the measurement was unaffected by color or turbidity, any type of blood sample could be analyzed: platelet-poor plasma, platelet-rich plasma, and whole blood. The test strips with the predried reagents were stable when stored for several months before testing. Analysis of the combined results obtained with different activators allowed discrimination between defects of the extrinsic, intrinsic, and common coagulation pathways. Activated protein C (APC) predried on the strips allowed identification of APC-resistance in plasma and whole blood samples. Conclusions: The biosensor system provides a new method for assessing thrombin generation in plasma or whole blood samples as small as 10 μL. The assay is easy to use, thus allowing it to be performed in a point-of-care setting.

Patients with Mild Hemophilia a with Discrepant FVIII Assays: Thrombin Generation and Bleeding Phenotype

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2844-2844 ◽  
Author(s):  
Maria Elisa Mancuso ◽  
Maria Rosaria Fasulo ◽  
Antonino Cannavò ◽  
Flora Peyvandi ◽  
Elena Santagostino
Keyword(s):  
Thrombin Generation ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Bleeding Tendency ◽  
Thrombin Generation Assay ◽  
Dental Procedures ◽  
Single Center Study ◽  
Study Population ◽  
Bleeding Score ◽  
Bleeding History

Abstract Introduction: Patients with mild hemophilia A (MHA) have factor VIII (FVIII) levels between 6 and 40%. FVIII clotting activity can be measured by one-stage (OS), two-stage (TS) and chromogenic substrate (CSA) assays. Discrepancy between assays was reported, raising the issue of which is the most adequate to make diagnosis and predict bleeding tendency. Thrombin generation assay (TGA) was investigated as a tool to discriminate bleeding phenotype in patients with hemophilia. Aim: This single-center study was aimed at ascertaining the prevalence of discrepant cases among patients with MHA and at correlating FVIII and TGA results with the bleeding phenotype. Methods and Results: 134 consecutive MHA patients, aged 1-81 years (median 40) and diagnosed on the basis of FVIII:OS >5% were recruited between March 2012 and April 2013 during outpatient follow-up visits. Patients who had developed anti-FVIII inhibitors were excluded. Data on bleeding history were collected from medical files. At recruitment, the bleeding score was assessed and blood drawn in a non-bleeding state to measure FVIII:OS, FVIII:CSA and TGA. Genetic analysis was performed to ascertain FVIII gene defects. Thirty-six patients (26%) aged 2-76 years (median 40) had assay discrepancy being the FVIII:OS/CSA ratio <0.5 in 2 and >2.0 in 34. The former were brothers aged 27 and 34 years, with Arg1689His mutation and similar FVIII and TGA values (FVIII:OS 31 and 34%; FVIII:CSA 70 and 85%; ETP 1629 and 1475 nMxmin; peak 163 and 173 nM). The youngest bled after tooth extraction and had 2 post-traumatic haemorrhages, while the oldest never bled despite surgery/dental procedures. Among the 34 patients with FVIII:OS/CSA >2.0, 6 (18%) had FVIII:OS >40% despite a bleeding history and 2 (6%) never bled; the latter belong to a triplet of brothers, all with similar FVIII and TGA values (FVIII:OS 24-28%; FVIII:CSA 9% in all; ETP 1447-1496 nMxmin; peak 106-116 nM). Among 98 non-discrepant patients aged 1-81 years (median 40), the median FVIII:OS/CSA ratio was 1,45 (IQR: 1,26-1,65) and only 4 (4%) never bled. Major features of patients with FVIII:OS/CSA ratio >2.0 (from 26 families) and non-discrepant patients (from 90 families) are shown in the table. Three families contributed to both groups. Conclusions: In this series of MHA patients, one third were discrepant and 20% of them would not have been diagnosed by FVIII:OS. TGA values were consistent with FVIII:CSA and concordant with the bleeding score. However these potentially misdiagnosed patients represented a minority of the whole study population. TablePatients with FVIII:OS/CSA ratio >2.0 (n=34)Non-discrepant patients (n=98)Median FVIII:OS, % (range)20 (7-58)18 (5-61)Median FVIII:CSA, % (range)8 (2-25)12 (4-37)Median ETP, nMxmin (IQR)1475 (1278-1646)1373 (1117-1610)Median Peak, nM (IQR)116 (84-150)131 (103-168)Median bleeding score (IQR)3 (2-6)4 (2-5) Disclosures No relevant conflicts of interest to declare.

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.

scholarly journals Expression and Release of Platelet Protein Disulfide (PDI) Isomerase Is Increased in Patients with Hemophilia a

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1085-1085
Author(s):  
Florian Langer ◽  
Minna Voigtländer ◽  
Katharina Holstein ◽  
Brigitte Spath ◽  
Walter Fiedler ◽  
...  
Keyword(s):  
Plasma Levels ◽  
Research Funding ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Antigen Expression ◽  
Compensatory Mechanism ◽  
Bleeding Tendency ◽  
Fibrin Deposition ◽  
Significant Difference ◽  
Support Research

Abstract Hemophilia A is an X-linked, recessive bleeding disorder caused by congenital factor VIII (FVIII) deficiency. Although the bleeding tendency largely depends on residual FVIII activity (FVIII:C), there is tremendous heterogeneity in bleeding frequency and severity among individuals with similar FVIII:C plasma levels. It is therefore likely that additional factors modulate thrombin generation and fibrin deposition in patients with hemophilia A. PDI is an abundant oxidoreductase with chaperone activity that is also present in human platelets and released upon activation. Preclinical studies indicate that extracellular PDI is critical to hemostasis, thrombosis and vascular inflammation. In particular, PDI has been implicated in monocyte/macrophage tissue factor activation, integrin regulation and platelet-associated thrombin generation. Furthermore, impaired PDI release has most recently been shown to contribute to the bleeding tendency of Hermansky-Pudlak syndrome, an inherited platelet function defect. To explore the role of platelet PDI in hemophilia A, we studied 24 patients (15 severely, 5 moderately and 4 mildly affected) in comparison to 12 age- and sex-matched controls. Expression of PDI antigen on resting platelets and platelets stimulated with either 20 µM ADP or 50 µM thrombin receptor activator peptide 6 (TRAP-6) was assessed by flow cytometry using a fluorescently labeled monoclonal antibody. Analysis of CD41 and CD62P (P-selectin) served as positive controls for constitutive platelet antigen expression and α-granule secretion, respectively. In addition, release of soluble PDI antigen into platelet supernatants was measured by ELISA. There was no significant difference in baseline CD41, CD62P and PDI antigen expression between patients and controls. Furthermore, ADP- and TRAP-6-induced CD62P expression was similar between the two groups (percent positive platelets in patients vs. controls: 28±14 vs. 32±15% and 80±12 vs. 83±9% for ADP- and TRAP-6-treated platelets, respectively). However, expression of PDI antigen on platelets stimulated with either ADP (3.3±2.1 vs. 1.5±1.2%, P<0.01) or TRAP-6 (3.4±1.7 vs. 2.1±1.3%, P<0.05) was significantly increased in patients compared to controls. While ADP-induced release of PDI antigen into platelet supernatants was similar between the two groups and not significantly different from baseline, stimulation with TRAP-6 resulted in significantly increased PDI antigen levels in platelet releasates from patients vs. controls (median, range): 1.5, 0.2-23.2 ng/mL vs. 0.4, 0.2-1.9 ng/mL (P<0.01). Importantly, in two patients with exceedingly high TRAP-6-induced PDI release over baseline (4.8 vs. 0.3 ng/mL and 23.2 vs. 2.8 ng/mL), findings were consistent when platelets were isolated and stimulated on a separate occasion (5.5 vs. 1.3 ng/mL and 10.2 vs. 0.2 ng/mL). Taken together, agonist-induced platelet PDI expression was significantly increased in patients with congenital hemophilia A. Furthermore, release of PDI antigen into supernatants of TRAP-6-activated platelets was significantly increased in patients compared to healthy controls. Up-regulation of platelet PDI may thus represent a compensatory mechanism under conditions of defective thrombin generation and fibrin deposition, and variations in platelet PDI expression and release could at least partially explain the heterogeneity in bleeding severity among patients with congenital hemophilia A and similar FVIII:C plasma levels. Disclosures Langer: Baxalta: Consultancy, Other: Travel support; Pfizer: Research Funding; CSL Behring: Consultancy, Other: Travel support, Research Funding. Voigtländer:CSL Behring: Other: Travel support. Holstein:CSL Behring: Consultancy, Other: Travel support, Research Funding.

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.

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