Thrombelastographic Analysis Research Articles

Coagulation disorders in SARS-CoV-2 infection.

A better understanding of the pathogenetic mechanisms triggered by SARS-CoV-2 infection may contribute to a more effective management of patients with COVID-19. Coagulation dysfunction is a key pathogenetic element of this disease as well as a challenge for practitioners. Marked inflammatory process found in severe forms of COVID-19, the complement activation, the cytokine storm, and disruption of the renin-angiotensin-aldosterone system are involved in the onset of thrombotic microangiopathy and large vessel coagulopathy. Virus-induced procoagulant activity occurs at the systemic level. Intravascular microthrombi disrupt vascularization in various tissues and organs, contributing to the occurrence of multiorgan failure and explain the higher morbidity and all-cause mortality of patients. It is estimated that almost 20% of patients with COVID-19 have significant coagulation disorders, and about a quarter of those hospitalized in intensive care units are prone to develop thrombosis events under prophylactic anticoagulant treatment. Some of patients who have been immunized after healing from the SARS-CoV-2 infection have a hypercoagulable state and are prone to develop thrombosis. Hypercoagulability is supported by thrombelastographic analysis: patients have an acceleration of the propagation phase of blood clot formation and higher clot strength. Markers of coagulation dysfunction in SARS-CoV2 are: decreased platelet count, increased INR, presence of fibrin degradation products, and especially higher plasma levels of D-dimers, which predict unfavorable outcome in these patients. Age, pre-existing diseases and associated risk factors, together with careful monitoring of clinical evolution and laboratory parameters allow the choice of the best personalized prophylactic or curative anticoagulant treatment.

Potency Equated Porcine and Bovine Mucosal Heparin Are Bioequivalent in Terms of Biochemical and Pharmacological Effects

Introduction: Currently, there is a shortage of porcine heparin due to limited availability of porcine mucosa and supply chain issues. Bovine heparin has been used for clinical purposes globally and is being considered for reintroduction in the U.S. On a mass basis, commercially available porcine heparins exhibit a higher potency (180-220 units/mg) than their bovine counterpart (130-150 units/mg). Therefore, at gravimetric levels, the porcine heparins exhibit stronger biochemical and pharmacological effects in comparison to bovine heparin. Since heparin is standardized in biologic units relative to the USP or EP Standard, it is hypothesized that potency equated porcine and bovine heparin will exhibit similar biologic activities in laboratory assays. The purpose of this study was to compare the biologic properties of the porcine and bovine heparin at USP potency equated levels in standardized laboratory assays used for measuring and monitoring heparins. Materials and Methods: Active pharmaceutical ingredient (API) porcine mucosal heparin (200 units/mg) of U.S. origin was obtained from Medefil Inc. (Glendale Heights, IL). Bovine heparin (140 units/mg) was obtained from KinMaster Produtos Químicos (Passo Fundo, Brazil). Whole blood Activated Clotting Time (ACT) and Thrombelastographic analyses were performed. Clot based assays such as aPTT, TT, and prothrombinase induced clotting time (PiCT) were performed using citrated human plasma supplemented with heparins up to 1 anti-Xa unit/mL. Anti-Xa and anti-IIa activities were determined using chromogenic antiprotease assays (Biophen Kits). In a purified antithrombin-supplemented system, the inhibitory effects of these agents were measured in terms of anti-Xa and anti-IIa activities and expressed as IC50 values. Thrombin generation inhibition was measured using a kinetic assay (CAT system, Diagnostica Stago, Paris, France). Protamine and platelet factor 4 neutralization profiles were investigated in plasma-based systems. The heparin induced thrombocytopenia antibody (HIT) interaction profile was assessed using platelet aggregometry. Results: Both heparins produced comparable prolongation of the ACT to a range of 200-250 seconds. The thrombelastographic profile was comparable at concentrations of 0.125 unit/mL and 2.5 units/mL. Whole blood and plasma clotting times (aPTT and TT assays) were comparable at concentrations up to 1 unit/mL. In the chromogenic anti-Xa and anti-IIa assays, the behavior of both agents was also comparable. In the purified system, the IC50 values for both agents for the anti-Xa activity ranged from 0.33-0.40 unit/mL, whereas the anti-IIa values ranged from 0.38-0.42 unit/mL; no significant differences were noted between the porcine and bovine heparins. In the thrombin generation assays, in terms of peak thrombin generation, area under the curve, and lag time, both the porcine and bovine heparins showed comparable effects. The protamine neutralization profiles of the porcine and bovine heparin exhibited variable assay dependent results. Potency adjusted bovine heparin required higher amount of protamine for the complete neutralization of the biologic effects in comparison to the porcine heparin. Similar results were obtained in the platelet factor 4 neutralization studies. In the functional HIT screening assay, both heparins exhibited a similar concentration dependent aggregation of human platelets. These results are summarized in the Table. Summary and Conclusion: These results show that at potency adjusted concentrations, porcine and bovine heparin exhibit comparable biochemical and anticoagulant responses in whole blood, plasma based, and purified biochemical assays. Most notably, both the porcine and bovine heparin produced comparable HIT antibody mediated aggregation responses. Thus, the proposed approach to standardize heparins against a common standard using a biologic assay such as the USP method is valid. These results warrant additional validation studies to reintroduce bovine heparin for clinical use. Table Disclosures Jeske: KinMaster Produtos Quimicas: Research Funding.

Association of Thrombelastographic Parameters with Complications in Patients with Intracranial Aneurysm After Stent Placement

A prospective trial was conducted to investigate the platelet function and association between thrombelastographic (TEG) parameters and embolic or hemorrhagic complications in patients with intracranial aneurysm undergoing stent treatment. Between September 2013 and June 2016, we prospectively recruited patients with intracranial aneurysm who were treated with stent-assisted coiling. TEG parameters were used to assess the platelet function before stenting procedures. The primary study end point was the onset of ischemic stroke, transient ischemic attack, or silent ischemic events in the territory of the stented artery within 6 months after the procedure. The secondary end point was assessed by bleeding events. Four hundred and thirty-one patients with 453 intracranial aneurysms were enrolled. A total of 519 neurovascular stents were implanted. During the follow-up, a total of 70 primary end points (16.2%) and 59 secondary end points (13.7%) were detected. Thromboembolic complications such as symptomatic and slient ischemic complications were more frequently observed in patients with large aneurysms (>10 mm, P= 0.01), lower adenosine diphosphate (ADP) inhibition rate (P < 0.0001), and higher ADP-induced platelet-fibrin clot strength (maximum amplitude of adenosine diphosphate [MA-ADP]) (P < 0.0001). Besides, based on multivariate analysis, a higher ADP inhibition ratio was identified as a significant independent predictor of subsequent bleeding events (P < 0.0001). According to the receiver operating characteristic curve analysis, the safe range of the ADP inhibition ratio and MA-ADP of the TEG analysis were identified as 29.45%-55.4% and <46.15, respectively. The ADP inhibition ratio and MA-ADP of TEG analysis were associated with subsequent cerebral ischemic events and intracranial or extracranial bleeding events in patients with intracranial aneurysm after stent treatment.

Iron and carbon monoxide attenuate Crotalus atrox venom-enhanced tissue-type plasminogen activator-initiated fibrinolysis.

In addition to degrading fibrinogen as a source of consumptive coagulopathy, rattlesnake venom has also been demonstrated to enhance fibrinolysis and degrade alpha-2-antiplasmin. The goals of this investigation was to characterize the kinetic fibrinolytic profile of Crotalus atrox venom in the absence and presence of tissue-type plasminogen activator (tPA), and to also ascertain if iron and carbon monoxide (CO, a positive modulator of alpha-2-antiplasmin) could attenuate venom-enhanced fibrinolysis. Utilizing thrombelastographic methods, the coagulation and fibrinolytic kinetic profiles of human plasma exposed to C. atrox venom (0-2 μg/ml) were determined in the absence or presence of tPA (0-100 IU/ml). Then, either separately or in combination, plasma was exposed to iron (ferric chloride, 10 μmol/l) or CO (carbon monoxide-releasing molecule-2, 100 μmol/l) prior to incubation with venom; the plasma sample was subsequently subjected to thrombelastographic analysis with addition of tPA. Venom exposure in the absence of tPA did not result in detectable fibrinolysis. In the presence of tPA, venom markedly enhanced fibrinolysis. Iron and CO, markedly attenuated venom enhancement of fibrinolysis. C. atrox venom enhances tPA-mediated fibrinolysis, and interventions that enhance/protect alpha-2-antiplasmin activity significantly attenuate venom-enhanced fibrinolysis. Future preclinical investigation is required to determine if iron and CO can attenuate venom-mediated degradation of alpha-2-antiplasmin-dependent fibrinolytic resistance.

Effect of Recombinant Lubricin on Coagulation Parameters in Human Blood

Lubricin (proteoglycan 4; PRG4) is a proteoglycan which acts as a joint lubricant. Lubricin is present in synovial fluid and plays a major role in synovial homeostasis. A recombinant form of lubricin, rhPRG‐4 is developed to treat a variety of inflammatory diseases. (Lubris Pharmaceutical). The purpose of this study is to determine the effect of rhPRG‐4 on blood coagulation utilizing various experimental studies.rhPRG‐4 was supplemented with citrated whole blood from normal human donors (n = 10) to obtain concenration range of 0 ‐ 200 mg/mL. Activated clotting time (ACT), thrombelastographic analysis (TEG), and whole blood prothrombin time (PT), partial thromboplastin time (aPTT), and thrombin times (TT) were measured. Similarly, citrated plasma was supplemented with rhPRG‐4 at 0‐200 mg/ML to determine PT, aPTT and TT. The effect of this agent was also studied on the generation of proteases. In addition, rhPRG‐4 was also studied for its effect on platelet aggregation.rhPRG‐4 did not produce any effect on the whole blood clotting assays, such as the PT, aPTT and TT. Similarly, in the citrated plasma, none of the clotting assays were altered. rhPRG‐4 did not produce any inhibition of thrombin generation in plasma. Furthermore, it did not produce any inhibition or augmentation of agonist‐induced platelet aggregation. These studies suggest that rhPRG‐4 does not modify whole blood and plasma coagulation profile and produce and more relation of platelet function.These studies suggest that because of no potential alteration of the coagulation system, lubricin is not expected to modulate bleeding or thrombotic processes in clinical indications.

Tobacco smoke-induced hypercoagulation in human plasma

Virtually every disease state associated with chronic or acute thrombosis has had smoking identified as a risk factor. Further, smoking enhances clot strength as assessed by thrombelastography. Critically, carbon monoxide, a product of cigarette smoking, has been demonstrated to enhance plasmatic coagulation in vitro via modulation of a heme associated with fibrinogen. We hypothesized that plasmatic hypercoagulability and formation of carboxyhemefibrinogen (COHF) detected with thrombelastographic methods would be observed after cigarette smoking. Smoking participants (n = 20, two cigarettes consumed within 90 min, average carboxyhemoglobin concentration of 5%) had plasma collected and normal participant (n = 20) plasma was also obtained. Thrombelastographic analyses revealed that plasma obtained from smokers had an 86% greater velocity of clot growth and 65% larger clot strength than normal participant plasma. Forty-five percent of smokers had plasma clot strength that exceeded the 95th percentile of normal participant plasma values; 45% of smoking participants had detectable COHF; and 20% of smoking participants were both hypercoagulable with COHF present. We conclude that smoking induced a hypercoagulable state and COHF formation in an important portion of participants tested. Future investigations of the effects of smoking, plasmatic hypercoagulation and COHF formation are planned in populations with established atherosclerotic/thrombotic disease.

Can divergent plasmin–antiplasmin–carbon monoxide interactions in young, healthy tobacco smokers explain the ‘smokerʼs paradox’?

In the setting of acute myocardial infarction, decreases in early/late mortality, reocclusion after thrombolysis, and restenosis rate after percutaneous intervention are lower in smokers - this phenomenon has been designated as the 'smoker's paradox'. These benefits of smoking, however, are abrogated by stent placement. We hypothesized that fibrinolytic vulnerability would change in response to smoking, and that inhaled carbon monoxide may play a role. Smoking patients (n = 20, two cigarettes consumed within 90 min, average carboxyhemoglobin concentration of 5%) had plasma collected and normal individual (n = 20) plasma was also obtained. Thrombelastographic analyses conducted with addition of tissue-type plasminogen activator revealed that with the exception of the rate of thrombus generation, there was little difference in fibrinolytic kinetics between normal and smoking individuals. Addition of exogenous carbon monoxide resulted in diminished fibrinolytic response to the same extent in both groups. Subanalyses demonstrated that the smoking cohort had both hyperfibrinolytic and hypofibrinolytic patients as defined by confidence interval (5-95%) values generated from normal individuals. Addition of carbon monoxide reduced hyperfibrinolytic parameter values by 80% in smokers, whereas only a 17% decrease in hypofibrinolytic values changed. Our investigation suggests that 'smoker's paradox' involves a marked change in the character of the plasmin-antiplasmin-carbon monoxide interaction. Further investigation will be required to further define the molecular mechanism responsible for the 'smoker's paradox'.

Influence of chronic administration of anabolic androgenic steroids and taurine on haemostasis profile in rats

Anabolic androgenic steroids (AAS) are synthetic derivatives of testosterone with thrombogenic potential in high doses and long-term administration. Taurine, a widely distributed amino-sulfonic acid, is known for its beneficial effects in hypercoagulable states. In order to assess the impact of chronic administration of high doses of AAS and taurine upon haemostasis process in rats, 40 male Wistar rats were divided into four equal groups: control group (group C) - no treatment; androgen group (group A) - received 10 mg/kg per week of nandrolone decanoate (DECA); taurine (group T) - received oral supplementation of 2% taurine in drinking water; androgen and taurine group (group AT) - concomitant administration of DECA and taurine. After 12 weeks, blood samples were collected and haemostasis parameters were assessed with the thrombelastographic (TEG) analysis system: reaction time, clot kinetics (K, α), final clot strength, coagulation index and the clot lysis (Ly30). Nandrolone significantly decreased reaction time in group A compared with control (P<0.001), whereas taurine significantly increase reaction time (P=0.01), and this effect was maintained in group AT compared with group A (P=0.009). Similar differences between groups have been recorded for the clot kinetics parameters K, α. The final clot strength and coagulation index were significantly increased in group A versus group C (P=0.04, respectively P<0.001), but not in group AT versus group C (P>0.05). There were no differences in clot lysis, as shown by Ly30. Nandrolone produces an accelerated clot development and an increased clot firmness in Wistar rats. Taurine association ensures a protective effect against this hypercoagulable state, partially restoring the altered parameters of the coagulation profile.

Freezing does not decrease carbon monoxide-mediated hypercoagulation and hypofibrinolysis in human plasma

Carbon monoxide (CO) has been demonstrated to enhance coagulation and attenuate fibrinolysis in vitro and in vivo. Hemostasis is affected by CO interactions with key heme-modulated molecules. We wished to determine whether freezing would affect CO-mediated changes in coagulation/fibrinolysis in plasma in anticipation of collecting samples both within our institution and from collaborating centers. Plasma was exposed to CO by addition of 0-100 μmol/l tricarbonyldichlororuthenium (II) dimer, with a portion of plasma immediately frozen at -80 °C. Unfrozen plasma was subjected to thrombelastographic analysis following tissue factor activation, with some samples exposed to tissue type plasminogen activator. Frozen plasma was subsequently thawed and similarly analyzed. Freezing did not significantly change CO-mediated enhancement of coagulation or attenuation of fibrinolysis. Hemostatic changes in plasma exposed to CO are not affected by a freeze-thaw cycle, which will permit local batch processing of samples and transport of samples on dry ice from collaborating centers.

Thrombelastographic Demonstration of Nitrite Inhibition of Platelet Function

Abstract 5148We have recently shown that nitrite ions in blood will inhibit platelet aggregation and activation as measured by aggregometry and flow cytometric analysis of P-selectin under partially deoxygenated conditions. We now show that these phenomena can be measured by thrombelastographic analysis, an alternative method which supplies additional parameters of clotting, using whole blood from healthy donors. Nitric oxide (NO) can be generated by endogenous nitric oxide synthase (NOS) as well as by a serial reduction pathway in which nitrate and nitrite are converted to NO via several enzymatic and nonenzymatic mechanisms. We have previously shown that nitrite anions at 0. 1μM inhibit platelet activation and aggregation in the presence of red blood cells through their reduction to NO and this effect was enhanced by deoxygenation. In the present study, we examined the influence of nitrite on the clotting properties of blood to investigate how nitrite may affect overall clotting processes by modulating platelet function. We measured major clotting parameters such as reaction time (R, minute of initial fibrin formation), angle (velocity of clot growth), and maximum amplitude (MA, clot strength) using thrombelastograph hemostasis analyzer (TEG5000®) in whole blood diluted with plasma to yield 20% Hct. Both the NO donor (DEANONOate) and nitrite showed inhibitory effects on clotting parameters resulting in delayed R, decreased angle, and reduced MA in a dose dependent manner. However, the sources of NO did not change any TEG parameters in plasma. These results suggest that this new clinical method may be used to follow NO inhibition of platelet-induced blood clotting and that the physiological effect of factors which determine NO bioavailability, such as endogenous levels of blood and tissue nitrite, could be used as biomarkers for predicting hemostatic potential. Disclosures:Schechter:NIH: Employment, co-inventor of NIH patent on use of nitrite in disease, co-inventor of NIH patent on use of nitrite in disease Patents & Royalties.

Comparison of citrated native and kaolin‐activated samples for thrombelastographic analysis in healthy dogs

Thrombelastographic (TEG) analysis is a test of global hemostasis in veterinary medicine; however, there have been limited comparisons of analysis of citrated native and kaolin-activated samples. The purpose of this study was to determine the variation in TEG variables between citrated native and kaolin-activated whole blood samples and to establish reference intervals for both sample types. Citrated whole blood samples were obtained from 40 healthy dogs. Thirty minutes after collection, TEG analysis was performed simultaneously on samples with and without kaolin-activation. Reaction time (R), clotting time (K), angle (α), maximum amplitude (MA), global clot strength (G), and clot lysis at 30 minutes (LY30) were recorded, and the concordance correlation coefficient (ρ(c)) was calculated for each sample type. Significant differences between results obtained for kaolin-activated and native samples were obtained for R (mean difference -1.3 minute, P = .0009), K (-0.7 minute, P = .0003), α (+5.1º, P = .002), MA (+2.4 mm, P = .002), and G (+568 dyn/cm(2), P = .0009). LY30 was not different between methods. There was substantial agreement between methods for G (ρ(c) = .69) and MA (ρ(c) = .65), moderate agreement for R (ρ(c) = .45) and α (ρ(c) = .44), fair agreement for K (ρ(c) = .29), and slight agreement for LY30 (ρ(c) = .04). The TEG variables were significantly altered by kaolin activation; however, some agreement between sample types suggests a consistent bias. In citrated whole blood activated with kaolin, clot formation time is shortened and the amplitude of the tracing is increased, resulting in a TEG tracing that appears to indicate relative hypercoagulability compared with that obtained using native citrated whole blood samples.

Equally increased hypercoagulability irrespective of using minimized or conventional ECC systems

Objective. Minimized extracorporeal circulation systems in coronary artery bypass may have less impairing effect on hematological parameters and bleeding compared to conventional systems. The aim of this study was to investigate whether the use of mini systems does result in an increased postoperative hypercoagulative status. Methods. Patients with increased risk of postoperative blood transfusion were randomly allocated to coronary bypass surgery using conventional or minimized extracorporeal circulation. Serial thrombelastographic analysis was performed preoperatively, at day 1 and 5. Results. Forty-six patients were included. In 56% of the patients in the experimental and in 62% in the control group an activated coagulation system, measured as maximal amplitude above normal reference was found preoperatively. This was normalized the day after operation. At day 5 a significant number of patients in the experimental group (p = 0.03) overshooted the preoperative maximal clot strength compared to the control group (p = 0.15); however, a direct intergroup analysis showed no difference. In both groups, hemoglobin levels and platelet counts decreased at day one after surgery. At day 5, hemoglobin was partly and platelets completely normalized. No differences were found with respect to transfusion needs. Conclusion. Mini systems seem to induce at least an equal increased state of hypercoagulability after coronary surgery. No clinical relevant differences could be demonstrated. This could indicate that patients after using mini systems are not protected against a postoperative state of increased hypercoagulability.

Effects of delayed anticoagulation and use of evacuated tubes on non‐activated thrombelastography in dogs

The effects of delayed anticoagulation and use of evacuated vacuum tubes in the collection of whole blood for nonactivated thrombelastography (TEG) are not known. The objective of this study was to examine the effects of delayed anticoagulation and use of vacuum-assisted blood collection tubes on results of nonactivated TEG. Twelve clinically healthy adult dogs were used in each of 3 studies. For each study, nonactivated TEG results from paired blood samples were compared. In study 1, the effect of delayed citrate anticoagulation was evaluated by collecting samples either into syringes containing citrate or into empty syringes followed by transfer to nonevacuated tubes containing citrate. In study 2, the effect of vacuum assistance in blood transfer was evaluated by collecting samples into syringes containing citrate and transferring either to nonevacuated plastic tubes or to evacuated plastic tubes. In study 3, the combined effects of delayed anticoagulation and vacuum assistance in blood transfer were evaluated by collecting samples into syringes containing citrate or into empty syringes followed by transfer to evacuated tubes containing citrate. Thrombelastographic analysis was performed in duplicate at 39°C after a 40-minute rest period. The collection methods that delayed anticoagulation and/or used evacuated tubes yielded samples that appeared more coagulable compared with samples not exposed to delay or evacuated tubes. Methods by which samples are collected affect results of nonactivated TEG and should be considered when establishing reference intervals, interpreting results, and publishing TEG results.

Intravenous injection of autologous amniotic fluid induces transient thrombocytopenia in a gravid rabbit model of amniotic fluid embolism

Amniotic fluid embolism (AFE) is a rare but catastrophic complication of pregnancy characterized by severe hypotension, cardiovascular collapse, and massive consumptive coagulopathy. Several animal models of this syndrome have been proposed, but most have yielded inconclusive results. The objective of this study was to develop a suitable animal model of AFE. Twelve rabbits in late gestation (25 days) were used. Amniotic fluid was collected from the fetal amniotic sacs after laparotomy, and autologous fluid was injected into 6 rabbits via the left auricular vein. Six other rabbits received saline (control group). Blood pressure, platelet counts, and coagulation variables were measured at baseline and at various intervals for 60 minutes after injection. The in vitro effect of amniotic fluid on coagulation was assessed by thrombelastographic (TEG) analysis. Injection of amniotic fluid did not reproduce clinical signs of AFE and had no effect on activated partial thromboplastin time (aPTT), prothrombin time (PT), or Factor VIII activity. However, significant thrombocytopenia was observed 5 minutes after administration of amniotic fluid and resolved by 60 minutes. In vitro addition of amniotic fluid to blood resulted in accelerated clotting on TEG tracings. The syndrome of AFE was not reproduced in this rabbit model. However, injection of autologous amniotic fluid induced a transient and severe thrombocytopenia. Moreover, TEG analysis indicated that amniotic fluid could initiate the coagulation cascade. Other factors such as the presence of meconium in amniotic fluid may be needed to provoke more severe clinical signs.

Generic and Branded Versions of Argatroban Exhibit Differential Anticoagulant Effects In Whole Blood, Plasma Based Assays and Thrombin Generation Assays

AbstractAbstract 5129Several generic versions of argatroban) (Mitsubishi; Tokyo, Japan) have been introduced in Japan (Argaron, Gartban, Slovastan). In addition, other generic versions of argatroban are being considered by the European and North American regulatory bodies. While the generic versions of argatroban exhibit similar antithrombin potency (Ki values), because of the differential compositional variations their anticoagulant effects in whole blood systems may differ due to their cellular and plasmatic protein interactions. Branded and generic versions of argatroban may exhibit differential anticoagulant actions in the whole blood and plasma based assays due to their differential interactions with blood cells, platelets and plasma proteins. Three generic versions of argatroban that are commercially available in Japan namely Argaron, Gartban and Slovastan and a powdered version of generic argatroban (Lundbeck) were compared with the branded argatroban. Native whole blood thrombelastographic (TEG) analysis was carried out at 0.1 ug/mL, the Activated Clotting Time (ACT) assay was carried out in a concentration range of 0–10 ug/mL, and such coagulation tests as the PT/INR, aPTT, Heptest, and calcium thrombin time were performed. Plasma retrieved from the supplemented whole blood was also assayed. Ratios of the clotting time test values from whole blood and plasma were calculated. Retrieved plasma samples were also assayed in the thrombin generation assays (TGA). All of the different versions of argatroban produced a concentration dependent anticoagulant effect in the native whole blood TEG and ACT. In the TEG, while argatroban and Slovastan showed a similar effect, Gartban, Argaron and a powdered generic showed weaker effects. Argatroban was also different in the ACT assay. At a concentration of 5 ug/ml the ACTs were, Arg 340+15.2 secs, S 297+10.5 secs, G 292.0+19.1 secs and A 285.2+21.7 secs. In the citrated whole blood systems, all agents produced a concentration dependent anticoagulant effect; however, the generic versions produced a stronger anticoagulant effect in comparison to branded argatroban (p<0.001). In the PT assay at 5 ug/mL, argatroban showed 32 ± 3 sec vs 40–50 sec for the generic products. Similarly in the aPTT, Heptest and thrombin time tests argatroban was weaker than the generic products. Differences among generic versions were also evident. Similar results were obtained in the retrieved plasma, however the ratio of whole blood over plasma varied from product to product. The IC50 of the generic and branded argatrobans in the TGA were also different. These results show that while in the thrombin inhibition assays generic and branded argatroban may show similar effects, these agents exhibit assay dependent differences in the whole blood and plasma based assays. Such differences may be more evident in the in vivo studirs where endothelial cells and other interactions may contribute to product individuality. Therefore, based on the in vitro antiprotease assays, generic argatrobans may not be considered equivalent and require a multi-parametric study. Currently available generic argatrobans may not be equivalent in the in vivo anticoagulant effects. Therefore, clinical validation of the clinical equivalence for these drugs is warranted. Disclosures:No relevant conflicts of interest to declare.

The effects of in vitro hemodilution with 6% hydroxyethyl starch (HES) (130/0.4) solution on thrombelastograph analysis in patients undergoing liver transplantation

The aim of this study was to determine the effects of in vitro hemodilution with 6% HES (130/0.4) solution on thrombelastograph(®) (TEG) parameters in whole blood samples from patients with end-stage liver disease (ESLD). Enrollment consisted of 95 patients with ESLD undergoing liver transplantation. Blood was diluted by 11%, 22%, and 33% with 6% HES (130/0.4) solution. Normal saline was used as a control diluent. When blood was diluted by 33% with normal saline, only the reaction time (r) was increased (p < 0.0001) compared to the baseline value. When blood was diluted with 6% HES (130/0.4), 11% dilution decreased maximum amplitude (MA) (p = 0.003) compared to baseline. At 33% dilution, the r (p < 0.0001, vs. baseline) and K (p < 0.0001, vs. baseline; p < 0.0001, vs. normal saline) increased, and the MA, alpha angle, and coagulation index (p < 0.0001, vs. baseline; p < 0.0001, vs. normal saline) decreased. Hemodilution with 6% HES (130/0.4) solution results in TEG abnormalities even with 11% hemodilution, in whole blood samples of patients with ESLD undergoing liver transplantation.

Effect of Oxidized Fibrinogen on Hemostatic System: In Vitro Study

Standard coagulation assays were performed with control and oxidized fibrinogen (Fg), using prothrombin time (PT; 12.5 ± 0.4 vs 25 ± 0.8 seconds, P < .001) and activated partial thromboplastin time (aPTT; 33 ± 2.5 vs 63 ± 4.7 seconds, P < .001). Fibrin clot (MA), clot formation initiation (r), and rate of clot lysis (LY30) were measured, a reflection exposure of Fg to Fe(3+)/ ascorbate oxidative system by thrombelastograph (TEG) analysis (0, 6, 12, 24, and 48 hours, 6.2 ± 1.3 vs 5.5 ± 1.2, 4.3 ± 1.0 [P < .01], 3.9 ± 1.6, 3.2 ± 0.8, [P < .001]). Maximum amplitude level was found to be lower than control (69.1 ± 7.2 vs 67.9 ± 12.4, 64.0 ± 11.4, 60.2 ± 21.2, 42.2 ± 15.2, P < .001). The lysis rate was changed according to oxidation time between Fg exposed to Fe(3+)/ascorbate and control exposed to Fe( 3+)/ascorbate for the same treatment time (1.9 ± 0.71 vs 7 ± 0.5, 1.6 ± 0.1, 1.2 ± 0.5, 0.9 ± 1.3, P < .001). We revealed dysregulation of hemostatic system with contribution of oxidized Fg, which was in direct proportion to the intensity of Fg oxidation.

The effect of unfractionated heparin on thrombelastographic analysis in healthy dogs

To determine the effect of single and multiple doses of SQ heparin (200 U/kg) on the thrombelastogram of healthy dogs. Prospective study. University research facility. Six random-source female dogs. Baseline parameters, including a CBC with platelet count, prothrombin time, activated partial thromboplastin time (aPTT), and antithrombin were performed. Thrombelastography (TEG) and aPTT were performed hourly for 12 hours after unfractionated heparin dosing (200 U/kg, SQ). Anti-Xa activity was assayed at 0, 3, 6, and 8 hours. Heparin was then administered every 8 hours for 3 days. The sampling protocol on Day 4 was identical to Day 1. On Day 1, percentage change from baseline for TEG parameter R, as well as absolute values of K, angle, and maximum amplitude (MA) were evaluated. Statistically significant (P<0.01) prolongation of the R time and a decrease in angle and MA was seen in all dogs by hour 3. R and MA were unmeasurable for most dogs between 3 and 5 hours. All TEG tracings returned to baseline by 12 hours. Day 4 TEG tracings mimicked those on Day 1. Only 1 dog achieved aPTT values outside the reference interval on both days. Anti-Xa activity levels increased on Day 4 but not on Day 1. Based on post hoc in vitro analysis, prolongation of R time occurred at plasma heparin levels as low as 0.075 U/mL, well below the lower limit of detection of the anti-Xa activity level assay. Administration of SQ heparin results in progressive changes in the TEG tracing, with maximal change occurring 3-5 hours after dosing. The extensive prolongation of the R time also indicates that TEG may be too sensitive and limits its utility as a monitoring tool for unfractionated heparin therapy.

REVIEWS: Hemostatic resuscitation for massive bleeding: the paradigm of plasma and platelets—a review of the current literature

Continued hemorrhage remains a major contributor of mortality in massively transfused patients and controversy regarding the optimal management exists. Recent studies indicate a possible survival benefit in patients receiving a higher ratio of plasma and platelets (PLTs) to red blood cells (RBCs) than what is recommended in current transfusion guidelines. English databases were searched for reports of patients receiving massive transfusion that tested the effects of administration of plasma and/or PLTs in relation to RBCs on survival from January 1990 to March 2009. Fourteen retrospective studies involving 4594 patients were identified. Six tested the effect on survival in relation to fresh-frozen plasma (FFP)-to-RBC ratio, and five investigated FFP- and PLT-to-RBC ratios. Two studies evaluated implementation of massive transfusion protocols with preemptive FFP and PLT administration; one study based transfusion therapy on the result of the thrombelastograph (TEG) analysis versus historic controls. All studies reviewed demonstrate a survival benefit for patients who receive more FFP and PLT as part of the hemostatic resuscitation. When TEG was used to guide transfusion therapy an increase in FFP and PLT was also seen when compared to historic controls and this was associated with improved survival. High FFP- and PLT-to-RBC ratios seem to improve survival in patients with massive bleeding. Randomized studies evaluating TEG-guided transfusion therapy versus fixed ratios of plasma and PLTs to RBCs in massively bleeding patients is highly warranted.

Blood product ratio in acute traumatic coagulopathy - effect on mortality in a Scandinavian level 1 trauma centre

BackgroundTrauma is the leading cause of loss of life expectancy worldwide. In the most seriously injured patients, coagulopathy is often present on admission. Therefore, transfusion strategies to increase the ratio of plasma (FFP) and platelets (PLT) to red blood cells (RBC), simulating whole blood, have been introduced. Several studies report that higher ratios improve survival in massively bleeding patients. Here, the aim was to investigate the potential effect of increased FFP and PLT to RBC on mortality in trauma patients.MethodsIn a retrospective before and after study, all trauma patients primarily admitted to a level-one Trauma Centre, receiving blood transfusion, in 2001-3 (n = 97) and 2005-7 (n = 156), were included. In 2001-3, FFP and PLT were administered in accordance with the American Society of Anesthesiologists (ASA) guidelines whereas in 2005-7, Hemostatic Control Resuscitation (HCR) entailing pre-emptive use of FFP and PLT in transfusion packages during uncontrolled haemorrhage and thereafter guided by thrombelastograph (TEG) analysis was employed. The effect of transfusion therapy and coagulopathy on mortality was investigated.ResultsPatients included in the early and late period had comparable demography, injury severity score (ISS), admission hematology and coagulopathy (27% vs. 34% had APTT above normal). There was a significant change in blood transfusion practice with shorter time interval from admission to first transfusion (median time 3 min vs.28 min in massive bleeders, p < 0.001), transfusion of higher ratios of FFP:RBC, PLT:RBC and PLT:FFP in the HCR group but 30-day mortality remained comparable in the two periods. In the 2005-7 period, higher age, ISS and Activated Partial Thromboplastin Time (APTT) above normal were independent predictors of mortality whereas no association was fund between blood product ratios and mortality.ConclusionAggressive administration of FFP and PLT did not influence mortality in the present trauma population.