WHOLE BLOOD THROMBOELASTOGRAPHY IN HEALTHY ADULT CAMELIDS (VICUGNA PACOS AND CAMELUS DROMEDARIUS).

Thromboelastography (TEG) provides a global assessment of hemostasis and fibrinolysis and has broad applications to identify and monitor coagulation dysfunction in veterinary patients. Although alpacas are susceptible to a wide variety of coagulopathies, the assessment of TEG has not been reported in clinically healthy alpacas to date. The purpose of this study was to evaluate the analytical performance of recombinant human tissue factor (rhTF)- and kaolin-activated TEG and to establish reference intervals for TEG parameters (reaction [R] and clotting [K] times, angle [α], maximum amplitude [MA], and shear elastic modulus [G]) in healthy, adult alpacas. Kaolin and rhTF-activated TEG were performed using citrated whole blood samples from 20 clinically healthy, nonpregnant, adult Huacaya alpacas each after 30 min of sample storage at room temperature. Six individuals of a related species, dromedary camels, were also sampled for comparative purposes. All data were presented descriptively, assessed for normality, and compared using either independent-sample t tests or Mann-Whitney U tests, with P ≤ 0.05 considered significant. Reference intervals were calculated using a robust method and Box-Cox-transformed data. Mean TEG values (reference intervals) were determined for rhTF-activated TEG as follows: R 6.99 min (3.41-12.71), K 3.43 min (1.61-6.42), α 48.51° (27.21-67.38), MA 52.05 mm (21.53-65.92), and G 5.71 kdyn/cm2 (1.87-9.60), while mean values (reference intervals) for kaolin-activated TEG included R 7.72 min (4.48-11.43), K 4.24 min (2.03-9.20), α 45.06° (23.66-64.20), MA 52.18 mm (33.49-66.63), and G 5.78 kdyn/cm2 (NR-9.66). None of the measured TEG values differed significantly between activators, suggesting that activator choice may have a limited effect on TEG parameters in healthy alpacas. TEG results in alpacas were comparable to those of dromedary camels. These results will thus provide a useful starting point in the evaluation of hemostasis in adult camelids.

FVIII activity following FVIII protein infusion or FVIII gene transfer predicts the bleeding risk in hemophilia A rats.

BACKGROUND: Prophylactic replacement therapy in hemophilia A (HA) patients does not adequately prevent bleeds and arthropathic complications. A more refined understanding of the relationship between coagulation factor VIII (FVIII) levels and bleeding risk during protein prophylaxis, or with gene therapy, is needed to improve patient care. OBJECTIVES: Investigate this relationship in the HA rat, a model exhibiting spontaneous bleeds and development of arthropathy similar to HA patients. METHODS: Human B domain-deleted FVIII was delivered to HA rats via adeno-associated virus (AAV)-mediated gene transfer or multiple intravenous protein injections. RESULTS AND CONCLUSIONS: After 12 weeks of observation, both approaches significantly reduced bleeds per animal and increased the proportion of bleed-free animals compared with controls (43% vs 0%, respectively [AAV]; 75% vs 8%, respectively [injection]). Both approaches resulted in an anti-FVIII inhibitory response in 20% to 37% of treated animals, similar to HA patients. Inhibitory antibodies were refractory to clinical improvement (reduction of bleeds) only in the AAV-based prophylaxis. An integrated model-based analysis of data on FVIII exposure and bleeding events was performed. This predicted the bleeding risk at any given circulating FVIII activity. Specifically, 4.8 or 10 IU/dL FVIII (0.048 and 0.1 IU/mL, respectively) were predicted to reduce bleeding risk by 90% or 95%, respectively, compared with untreated controls. Our data establish the utility of the HA rat model in FVIII prophylaxis studies and describe how FVIII activity affects bleeding risk in this setting. These enable further studies on FVIII prophylaxis focusing on disease complications for an optimized treatment of HA patients.

Rotational thromboelastometry can predict the probability of bleeding events in a translational rat model of haemophilia A following gene-based FVIIa prophylaxis.

INTRODUCTION: Monitoring of clinical effectiveness of bypassing agents in haemophilia patients is hampered by the lack of validated laboratory assays. Thromboelastography (TEG) and rotational thromboelastometry (ROTEM) have been evaluated for predicting clinical effectiveness of bypassing agents, however, with limited success. AIM: Application of a longitudinal model-based approach may allow for a quantitative characterization of the link between ROTEM parameters and the probability of bleeding events. METHODS: We analyse longitudinal data from haemophilia A rats receiving gene-based FVIIa prophylaxis in terms of total circulatory levels of FVII/FVIIa, clotting time (CT) measured using ROTEM and the probability of bleeding events. RESULTS: Using population pharmacokinetic-pharmacodynamic (PKPD) modelling, a PK-CT-repeated time-to-event (RTTE) model was developed composed of three submodels (a) a FVII/FVIIa PK model, (b) a PK-CT model describing the relationship between predicted FVIIa expression and CT and (c) a RTTE model describing the probability of bleeding events as a function of CT. The developed PK-CT-RTTE model accurately described the vector dose-dependent plasma concentration-time profile of total FVII/FVIIa and the exposure-response relationship between AAV-derived FVIIa expression and CT. Importantly, the developed model accurately described the occurrence of bleeding events over time in a quantitative manner, revealing a linear relationship between predicted change from baseline CT and the probability of bleeding events. CONCLUSION: Using PK-CT-RTTE modelling, we demonstrated that ROTEM parameters can accurately predict the probability of bleeding events in a translational animal model of haemophilia A.

D-Dimer Concentrations and Thromboelastography in Five Dogs With Ischemic Stroke.

Ischemic stroke is a condition increasingly recognized in dogs; however, the number of publications on dogs with ischemic stroke is still limited and hemostatic parameters are infrequently reported. D-dimer levels have been shown to be elevated in people with acute ischemic stroke compared to a healthy control population and it has been proposed that a normal D-dimer can be used to exclude thromboembolism in dogs. In this case series, we report hemostatic parameters, including D-dimer and thromboelastography (TEG) along with clinical and imaging findings for five dogs diagnosed with ischemic stroke. All dogs had a normal D-dimer concentration on presentation. A hypercoagulable state was identified in two dogs based on the results of the TEG, and was suspected in the remaining three cases based on a shortened TEG clot reaction time. Based on the findings in the present cases, a D-dimer within the normal reference range does not seem an appropriate negative predictor for canine ischemic stroke. The demonstration of a possible hypercoagulable state, as identified by the TEG, is an interesting finding which should be explored further to help reveal predisposing hypercoagulable conditions in dogs with ischemic stroke.

Gene-based FVIIa prophylaxis modulates the spontaneous bleeding phenotype of hemophilia A rats.

A sizable proportion of hemophilia inhibitor patients fails immune tolerance induction and requires bypass agents for long-term bleed management. Recombinant human-activated coagulation Factor VII (rhFVIIa) is an on-demand bypass hemostatic agent for bleeds in hemophilia inhibitor patients. Prophylactic use of rhFVIIa may enable sustained hemostatic management of inhibitor patients, but the critical relationship of rhFVIIa circulating levels and clinical outcome in that setting remains unclear. To address this in vivo, we used the rat hemophilia A (HA) model that exhibits spontaneous bleeds and allows longitudinal studies with sufficient statistical power. We simulated activated Factor VII (FVIIa) prophylaxis by adeno-associated virus (AAV) gene transfer of a rat FVIIa transgene. Compared with naive HA animals, rat FVIIa continuous expression affected the overall observed bleeds, which were resolved with on-demand administration of recombinant rat FVIIa. Specifically, although 91% of naive animals exhibited bleeds, this was reduced to 83% and 33% in animals expressing less than 708 ng/mL (<14 nM) and at least 708 ng/mL (≥14 nM) rat FVIIa, respectively. No bleeds occurred in animals expressing higher than 1250 ng/mL (>25 nM). Rat FVIIa expression of at least 708 ng/mL was also sufficient to normalize the blood loss after a tail vein injury. Continuous, AAV-mediated rat FVIIa transgene expression had no apparent adverse effects in the hemostatic system of HA rats. This work establishes for the first time a dose dependency and threshold of circulating FVIIa antigen levels for reduction or complete elimination of bleeds in a setting of FVIIa-based HA prophylaxis.

Rapid inflammation and early degeneration of bone and cartilage revealed in a time-course study of induced haemarthrosis in haemophilic rats.

OBJECTIVES: Detailed knowledge of the sequential cell and tissue responses following haemarthrosis is important for a deep understanding of the pathological process initiated upon extensive bleeding into the joint causing haemophilic arthropathy (HA). The underlying pathobiology driving haemarthrosis towards HA has been difficult to establish in detail, although animal models have shed light on some processes. Previous studies have focused on a single or a few distant time points and often only characterizing one tissue type of the joint. The objective of this study was, therefore, to carefully map early onset of synovitis and HA following induced haemarthrosis. METHODS: One hundred and thirty haemophilia A rats were subjected to induced haemarthrosis or a sham procedure in full anaesthesia and euthanized from 30 min to 7 days after the procedure. Pathological changes of the joints were visualized using micro-computed tomography, histology and immunohistochemistry. RESULTS: Synovitis developed within 24 h and was dominated by myeloid cell infiltrations. Cartilage and bone pathology were evident as early as 48-96 h after haemarthrosis, and the pathology rapidly progressed with extensive periosteal bone formation and formation of subchondral cysts. CONCLUSION: Fast, extensive and simultaneous cartilage and bone degeneration developed shortly after haemarthrosis, as shown by the detailed mapping of the early pathogenesis of HA. The almost immediate loss of cartilage and the pathological bone turnover suggest a direct influence of blood on these processes and are unlikely to be attributed simply to an indirect effect of inflammation.

The influence of inflammation and hematocrit on clot strength in canine thromboelastographic hypercoagulability.

OBJECTIVE: To investigate parameters causing canine thromboelastographic hypercoagulability and to investigate whether thromboelastography (TEG) with Cytochalasin D (Cyt D) added is related to parameters of platelet activity. DESIGN: Prospective observational study on hemostatic and inflammatory parameters. Data were collected between November 2012 and July 2013. SETTING: University teaching hospital. ANIMALS: Twenty-eight dogs suffering from diseases predisposing to thrombosis and 19 clinically healthy dogs. Diseased dogs were enrolled if they fulfilled inclusion criteria regarding age, size, informed client consent, and obtained a diagnosis of a disease that has been associated with thrombosis or hypercoagulability. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Parameters of coagulation and anticoagulation, fibrinolysis, and antifibrinolysis, platelet activity, inflammation, platelet count, and hematocrit were measured using CBC, TEG, platelet aggregation on multiplate, platelet activity on flow cytometry, and hemostatic and inflammatory markers on plasma and serum analyses. ANOVA and multilinear regression analyses indicated that especially hematocrit and the inflammatory parameters C-reactive protein and interleukin-8 showed best association with overall clot strength in diseased dogs with hypercoagulable TEG tracings. Ratios presumed to reflect platelet contribution to the TEG tracing obtained in TEG analyses with Cyt D were related especially with hematocrit and P-selectin expression of platelets measured after γ-Thrombin activation on flow cytometry. CONCLUSION: Overall clot strength in TEG analyses of the hypercoagulable dogs included in the present study appears to be primarily associated with inflammation as well as hematocrit. Furthermore, the ratio between standard TEG analyses and TEG analyses with Cyt D may reflect some degree of platelet activity.

Acute Haemarthrosis in the Haemophilia A Rat Generates a Local and Systemic Proinflammatory Response.

Background Replacement therapy with coagulation factor VIII (FVIII) concurrent with bleeds (on-demand) in haemophilia A (HA) patients has been hypothesized to increase the risk for antidrug antibodies (inhibitors). A danger signal environment, characterized by tissue damage and inflammation at the site of a bleed, is thought to contribute to the anti-FVIII response. The nature of this inflammatory reaction is, however, not fully known, and new insights will be valuable for both managing inhibitors and understanding arthropathy development. Objective To characterize the inflammatory response, locally and systemically, during the first 24 hours following a joint bleed in the HA rat. Methods HA rats received a needle-induced knee joint bleed (n = 83) or a sham procedure (n = 41). Blood samples were collected at selected time points from 0 to 24 hours post injury/sham. Synovial fluid, intra-articular knee tissue and popliteal lymph nodes were collected at 24 hours. Cytokine/chemokine concentrations and gene expression were measured. Results Gene expression analysis revealed a rapid inflammatory response in the injured knees, accompanied by significantly increased levels of specific gene products in the synovial fluid; IL-1ß, TNFα, KC/GRO, IL-6, Eotaxin, MCP-1, MCP-3, MIP-1α, MIP-2, RANTES, A2M and AGP. Plasma analysis demonstrated significantly increased systemic levels of KC/GRO and IL-6 in injured rats already after 5 to 6 hours. Conclusion A rapid proinflammatory response, locally and systemically, characteristic of innate immunity, was demonstrated. Results reveal a more comprehensive inflammatory picture than previously shown, with resemblance to human haemophilic arthropathy, and with unique correlation between gene expression level, synovial concentration and plasma concentration in individual rats.

Pulmonary vasculature in dogs assessed by three-dimensional fractal analysis and chemometrics.

Fractal analysis of canine pulmonary vessels could allow quantification of their space-filling properties. Aims of this prospective, analytical, cross-sectional study were to describe methods for reconstructing three dimensional pulmonary arterial vascular trees from computed tomographic pulmonary angiogram, applying fractal analyses of these vascular trees in dogs with and without diseases that are known to predispose to thromboembolism, and testing the hypothesis that diseased dogs would have a different fractal dimension than healthy dogs. A total of 34 dogs were sampled. Based on computed tomographic pulmonary angiograms findings, dogs were divided in three groups: diseased with pulmonary thromboembolism (n = 7), diseased but without pulmonary thromboembolism (n = 21), and healthy (n = 6). An observer who was aware of group status created three-dimensional pulmonary artery vascular trees for each dog using a semiautomated segmentation technique. Vascular three-dimensional reconstructions were then evaluated using fractal analysis. Fractal dimensions were analyzed, by group, using analysis of variance and principal component analysis. Fractal dimensions were significantly different among the three groups taken together (P = 0.001), but not between the diseased dogs alone (P = 0.203). The principal component analysis showed a tendency of separation between healthy control and diseased groups, but not between groups of dogs with and without pulmonary thromboembolism. Findings indicated that computed tomographic pulmonary angiogram images can be used to reconstruct three-dimensional pulmonary arterial vascular trees in dogs and that fractal analysis of these three-dimensional vascular trees is a feasible method for quantifying the spatial relationships of pulmonary arteries. These methods could be applied in further research studies on pulmonary and vascular diseases in dogs.

Global measurement of coagulation in plasma from normal and haemophilia dogs using a novel modified thrombin generation test - Demonstrated in vitro and ex vivo.

INTRODUCTION: Canine models of severe haemophilia resemble their human equivalents both regarding clinical bleeding phenotype and response to treatment. Therefore pre-clinical studies in haemophilia dogs have allowed researchers to make valuable translational predictions regarding the potency and efficacy of new anti-haemophilia drugs (AHDs) in humans. To refine in vivo experiments and reduce number of animals, such translational studies are ideally preceded by in vitro prediction of compound efficacy using a plasma based global coagulation method. One such widely used method is the thrombin generation test (TGT). Unfortunately, commercially available TGTs are incapable of distinguishing between normal and haemophilia canine plasma, and therefore in vitro prediction using TGT has so far not been possible in canine plasma material. AIM: Establish a modified TGT capable of: 1) distinguishing between normal and haemophilia canine plasma, 2) monitoring correlation between canine plasma levels of coagulation factor VIII (FVIII) and IX (FIX) and thrombin generation, 3) assessing for agreement between compound activity and thrombin generation in ex vivo samples. METHODS: A modified TGT assay was established where coagulation was triggered using a commercially available activated partial thromboplastin time reagent. RESULTS: With the modified TGT a significant difference was observed in thrombin generation between normal and haemophilia canine plasma. A dose dependent thrombin generation was observed when assessing haemophilia A and B plasma spiked with dilution series of FVIII and FIX, respectively. Correlation between FVIII activity and thrombin generation was observed when analyzing samples from haemophilia A dogs dosed with canine FVIII. Limit of detection was 0.1% (v/v) FVIII or FIX. CONCLUSION: A novel modified TGT suitable for monitoring and prediction of replacement therapy efficacy in plasma from haemophilia A and B dogs was established.

Oral Tolerance Induction in Hemophilia B Dogs Fed with Transplastomic Lettuce.

Anti-drug antibodies in hemophilia patients substantially complicate treatment. Their elimination through immune tolerance induction (ITI) protocols poses enormous costs, and ITI is often ineffective for factor IX (FIX) inhibitors. Moreover, there is no prophylactic ITI protocol to prevent anti-drug antibody (ADA) formation. Using general immune suppression is problematic. To address this urgent unmet medical need, we delivered antigen bioencapsulated in plant cells to hemophilia B dogs. Commercial-scale production of CTB-FIX fusion expressed in lettuce chloroplasts was done in a hydroponic facility. CTB-FIX (∼1 mg/g) in lyophilized cells was stable with proper folding, disulfide bonds, and pentamer assembly after 30-month storage at ambient temperature. Robust suppression of immunoglobulin G (IgG)/inhibitor and IgE formation against intravenous FIX was observed in three of four hemophilia B dogs fed with lyophilized lettuce cells expressing CTB-FIX. No side effects were detected after feeding CTB-FIX-lyophilized plant cells for >300 days. Coagulation times were markedly shortened by intravenous FIX in orally tolerized treated dogs, in contrast to control dogs that formed high-titer antibodies to FIX. Commercial-scale production, stability, prolonged storage of lyophilized cells, and efficacy in tolerance induction in a large, non-rodent model of human disease offer a novel concept for oral tolerance and low-cost production and delivery of biopharmaceuticals.

PET Imaging of Tissue Factor in Pancreatic Cancer Using 64Cu-Labeled Active Site-Inhibited Factor VII.

UNLABELLED: Tissue factor (TF) is the main initiator of the extrinsic coagulation cascade. However, TF also plays an important role in cancer. TF expression has been reported in 53%-89% of all pancreatic adenocarcinomas, and the expression level of TF has in clinical studies correlated with advanced stage, increased microvessel density, metastasis, and poor overall survival. Imaging of TF expression is of clinical relevance as a prognostic biomarker and as a companion diagnostic for TF-directed therapies currently under clinical development. Factor VII (FVII) is the natural ligand to TF. The purpose of this study was to investigate the possibility of using active site-inhibited FVII (FVIIai) labeled with (64)Cu for PET imaging of TF expression. METHODS: FVIIai was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with (64)Cu ((64)Cu-NOTA-FVIIai). Longitudinal in vivo PET imaging was performed at 1, 4, 15, and 36 h after injection of (64)Cu-NOTA-FVIIai in mice with pancreatic adenocarcinomas (BxPC-3). The specificity of TF imaging with (64)Cu-NOTA-FVIIai was investigated in subcutaneous pancreatic tumor models with different levels of TF expression and in a competition experiment. In addition, imaging of orthotopic pancreatic tumors was performed using (64)Cu-NOTA-FVIIai and PET/MRI. In vivo imaging data were supported by ex vivo biodistribution, flow cytometry, and immunohistochemistry. RESULTS: Longitudinal PET imaging with (64)Cu-NOTA-FVIIai showed a tumor uptake of 2.3 ± 0.2, 3.7 ± 0.3, 3.4 ± 0.3, and 2.4 ± 0.3 percentage injected dose per gram at 1, 4, 15, and 36 h after injection, respectively. An increase in tumor-to-normal-tissue contrast was observed over the imaging time course. Competition with unlabeled FVIIai significantly (P < 0.001) reduced the tumor uptake. The tumor uptake observed in models with different TF expression levels was significantly different from each other (P < 0.001) and was in agreement with the TF level evaluated by TF immunohistochemistry staining. Orthotopic tumors were clearly visible on the PET/MR images, and the uptake of (64)Cu-NOTA-FVIIai was colocalized with viable tumor tissue. CONCLUSION: (64)Cu-NOTA-FVIIai is well suited for PET imaging of tumor TF expression, and imaging is capable of distinguishing the TF expression level of various pancreatic tumor models.

Thromboelastography in Selected Avian Species.

Currently available assay methods and reagents are not optimized for evaluating avian hemostasis; therefore, assessing avian coagulopathies is challenging. Recently, thromboelastography (TEG), which measures the viscoelastic properties of blood, has been used clinically in mammalian species to diagnose and characterize hemostatic disorders. To evaluate TEG in healthy individuals of 6 avian species, we modified existing mammalian TEG protocols to allow analysis of citrated, avian whole-blood samples collected from scarlet ibis (Eudocimus ruber) (n = 13), American flamingos ( Phoenicopterus ruber ) (n = 13), helmeted Guinea fowl ( Numida meleagris ) (n = 12), Amazon parrots (Amazona species) (n = 9), Humboldt penguins ( Spheniscus humboldti ) (n = 6), and domestic chickens (n = 16). Activated partial thromboplastin time, prothrombin time, and fibrinogen were measured as a means of comparison. Regardless of the mode of activation, clot formation in the species studied was markedly delayed compared with mammals. Because of prolonged reaction time (14.7-52.7 minutes) with kaolin and diluted tissue factor, undiluted human tissue factor was used in all avian samples because it provided the shortest reaction time. Species differed significantly in reaction time (P = .007), clotting rate (P < .001), rate of clot formation (α angle; P < .001), and maximum amplitude (P < .001) values, indicating that species-specific reference intervals are necessary. Based on these results, TEG with specific reference intervals could prove useful in evaluating avian hemostatic disorders.

Parenteral lipids and partial enteral nutrition affect hepatic lipid composition but have limited short term effects on formula-induced necrotizing enterocolitis in preterm piglets.

BACKGROUND & AIMS: Rapid transition from total parenteral nutrition (TPN) to enteral feeding is a risk factor for necrotizing enterocolitis (NEC) in preterm infants. We hypothesized that partial enteral nutrition with colostrum, increased proportion of n-3 polyunsaturated fatty acids (PUFA), or exclusion of lipid in TPN would affect short term NEC sensitivity and liver function. METHODS: Preterm piglets were fed for three days after birth: 1) TPN with a standard lipid emulsion (Nutriflex Lipid Plus, TPN control group, n = 19), 2) PN plus bovine colostrum as partial enteral nutrition (PN/COL, n = 18), 3) TPN with fish oil (FO) lipids (Omegaven, TPN/FO, n = 19), or 4) TPN with no lipid (TPN/NL, n = 22). After TPN, piglets were fed formula for two days before tissue collection. RESULTS: None of the treatments had consistent effect on NEC incidence (∼40-50% across all groups), intestinal morphology and function, relative to TPN. In the liver, there were no signs of steatosis but PN/COL decreased the n-6 PUFA levels, leading to higher n-3/n-6 ratio, GGT activity, and plasma cholesterol and albumin levels, relative to TPN (all p < 0.05). TPN/FO increased the hepatic n-3 levels and n-3/n-6 ratio. TPN/NL treatment led to decreased hepatic n-6 level, n-3/n-6 ratio and bilirubin, albumin and triglycerides, and lowered blood clotting strength (-30%, TPN/NL vs. TPN/COL, p < 0.05). CONCLUSION: Partial enteral nutrition with colostrum, increased n-3 PUFAs in TPN, or removal of lipid from the TPN, all affect hepatic lipids and proteins in preterm neonates. These effects do not translate into improved hepatic function or NEC resistance, at least not short term.

Hemostatic analysis of dogs naturally envenomed by the African puffadder (Bitis arietans) and snouted cobra (Naja annulifera).

OBJECTIVE: To investigate hemostatic changes in dogs envenomed by cytotoxic (African puffadder) and neurotoxic snakes (snouted cobra) using thromboelastography (TEG) and plasma-based coagulation assays. DESIGN: Prospective observational clinical study. SETTING: University teaching hospital. ANIMALS: Eighteen client-owned dogs; 9 envenomed by African puffadder (Bitis arietans) and 9 by snouted cobra (Naja annulifera). Ten healthy dogs served as controls. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Blood was collected at presentation and 24 hours post envenomation. Platelet count, TEG, prothrombin time, activated partial thromboplastin time (aPTT), antithrombin activity, and fibrinogen (Fib) and C-reactive protein (CRP) concentrations were measured. Outcomes were analyzed using linear mixed models at 5% significance. At presentation, R time was significantly prolonged in the puffadder group compared to the cobra (P = 0.01) and control groups (P = 0.05). Platelet count was significantly lower in the puffadder compared to the cobra (P = 0.04) and control groups (P = 0.001), respectively. Antithrombin activity was significantly decreased in the puffadder (P = 0.002) and cobra groups (P = 0.004) compared to the control group. Both prothrombin time and activated partial thromboplastin time were significantly prolonged in the cobra group compared to the control group (P = 0.03 for both). The TEG variables, maximum amplitude (MA) and G, were significantly increased 24 hours post envenomation in the puffadder group compared to their values at presentation (P = 0.05 for both). Fib and CRP concentrations were significantly increased 24 hours post envenomation in both snake-envenomed groups. CONCLUSIONS: Prolonged clot initiation was a common feature in puffadder-envenomed dogs at presentation and this was likely venom induced. Snouted cobra-envenomed dogs were normo- to hypercoagulable at presentation. Dogs from both puffadder and cobra groups progressed to a more hypercoagulable by 24 hours post envenomation, most likely due to marked inflammation as indicated by the increased Fib and CRP concentrations. TEG proved a sensitive tool for detecting abnormal hemostasis in snake-envenomed dogs.

Systematic evaluation of evidence on veterinary viscoelastic testing part 4: Definitions and data reporting.

OBJECTIVE: To systematically examine evidence surrounding definitions and reporting of data for viscoelastic testing in veterinary medicine. DESIGN: Standardized, systematic evaluation of the literature, categorization of relevant articles according to level of evidence and quality, and development of consensus on conclusions for application of the concepts to clinical practice. SETTING: Academic and referral veterinary medical centers. RESULTS: Databases searched included Medline, CAB abstracts, and Google Scholar. CONCLUSIONS: All 4 standard thromboelastography (TEG) and rotational thromboelastometry (ROTEM) variables should be universally reported, and the reporting of shear elastic modulus in addition to maximum amplitude (MA) is encouraged. There is insufficient evidence to support universal usage of the coagulation index at this time. The K value and clot formation time are the most variable of the 4 parameters, with alpha angle, MA, and maximum clot firmness generally the least variable. Individual studies should report sufficient data on patients and institutional controls to enable definitions of hypo- and hypercoagulability to be evaluated post-hoc, and it is recommended that all studies specifically report how these conditions were defined. In reporting data relating to fibrinolysis, the TEG variables LY30, LY60, CL30, CL60, and the ROTEM variables LI30, LI60, ML, LOT, and LT should be documented. Studies should report sufficient data on patients and controls to enable definitions of hyper- and hypofibrinolysis to be evaluated post-hoc, and we suggest that standard TEG/ROTEM assays may be unable to detect hypofibrinolysis in companion animals. We recommend that every center establish reference intervals, which are specific to either TEG or ROTEM. These reference intervals should be established using veterinary clinical pathology guidelines, standardized protocols, and a minimum of 40 healthy animals. There are currently insufficient data in companion animals to suggest a utility for Vcurve variables beyond that of standard TEG variables.

Systematic evaluation of evidence on veterinary viscoelastic testing part 2: Sample acquisition and handling.

OBJECTIVE: To examine systematically the evidence on sample acquisition and handling for the thrombo elastography (TEG) and rotational thromboelastometry (ROTEM) viscoelastic point of care instruments and to identify knowledge gaps. DESIGN: Six questions were considered, addressing sampling site, collection system, anticoagulant, collection procedure, and sample storage. Standardized, systematic evaluation of the literature was performed. Relevant articles were categorized according to level of evidence (LOE). Consensus was developed regarding conclusions for application of concepts to clinical practice. SETTING: Academic and referral veterinary medical centers. RESULTS: PubMed and CAB abstracts were searched. Eighteen papers were initially chosen; 5 of these papers applied to > 1 domain question. Three papers were used to address 2 questions each, and 2 papers were used to address 3 questions each. Most papers were judged LOE 3 (Good or Fair). Two of 5 papers were judged to be the same LOE each time they were used; 2 papers were judged to be LOE 3, Fair for 1 question and 3, Good for a second question; 1 paper used to address 3 questions was judged LOE 3, Good twice and 3, Fair once. Fourteen additional papers were evaluated post hoc during manuscript preparation. CONCLUSIONS: Jugular venipuncture is recommended, but samples from IV catheters can be used. Consistent technique is important for serial sampling, and standardized sampling protocols are recommended for individual centers performing TEG/ROTEM. There is insufficient evidence to recommend use of a specific blood collection system, although use of evacuated blood tubes and 21-Ga or larger needles is suggested. Use of 3.2% buffered sodium citrate in a strict 1:9 ratio of citrate to blood is suggested. Suggested tube draw order is discard/serum, followed by citrate, EDTA, and then heparin. Samples should be held at room temperature for 30 minutes prior to analysis.

Systematic evaluation of evidence on veterinary viscoelastic testing part 1: System comparability.

OBJECTIVE: To systematically examine the evidence on system comparability between the thromboelastography and the rotational thromboelastometry viscoelastic point-of-care instruments and to identify knowledge gaps. DESIGN: Standardized, systematic evaluation of the literature, categorization of relevant articles according to level of evidence and quality, and development of consensus on conclusions for application of the concepts to clinical practice. SETTING: Academic and referral veterinary medical centers. RESULTS: Medline via PubMed, CAB abstracts, and Google Scholar were searched. A total of 8 relevant articles were chosen, none were in support of the question, 1 was neutral to the question (level of evidence [LOE] 6, Poor), and 7 were in opposition to the question (LOE 3 Good; LOE 6 Good; LOE 6 Fair; LOE 6 Poor). CONCLUSIONS: Results from the 2 analyzers are not directly comparable and extrapolation of the results from one machine to the other should be avoided. Standardization of the preanalytical variables (eg, blood collection, holding time, and temperature during holding) is strongly recommended. It is recommended that each site create their own "site specific" reference values for each machine and that test samples be compared only to the standardized reference values established at that center. Start-up and consumable costs vary between countries and local comparisons should be performed. Decisions should be made based on the expected use of the machine and if multiple operators will be using it.

Partnership on Rotational ViscoElastic Test Standardization (PROVETS): evidence-based guidelines on rotational viscoelastic assays in veterinary medicine.

OBJECTIVE: To systematically examine the evidence relating to the performance of rotational viscoelastic testing in companion animals, to develop assay guidelines, and to identify knowledge gaps. DESIGN: Multiple questions were considered within 5 parent domains, specifically system comparability, sample handling, assay activation and test protocol, definitions and data reporting, and nonstandard assays. Standardized, systematic evaluation of the literature was performed. Relevant articles were categorized according to level of evidence and assessed for quality. Consensus was developed regarding conclusions for application of concepts to clinical practice. SETTING: Academic and referral veterinary medical centers. RESULTS: Databases searched included Medline, Commonwealth Agricultural Bureaux abstracts, and Google Scholar. Worksheets were prepared evaluating 28 questions across the 5 domains and generating 84 assay guidelines. CONCLUSIONS: Evidence-based guidelines for the performance of thromboelastography in companion animals were generated through this process. Some of these guidelines are well supported while others will benefit from additional evidence. Many knowledge gaps were identified and future work should be directed to address these gaps and to objectively evaluate the impact of these guidelines on assay comparability within and between centers.

Systematic evaluation of evidence on veterinary viscoelastic testing part 3: Assay activation and test protocol.

OBJECTIVE: To systematically examine the evidence on activating agents and test protocols for the thrombelastography (TEG) and rotational thrombelastometry (ROTEM) viscoelastic point-of-care instruments and to identify knowledge gaps. DESIGN: Ten questions were considered, the primary question addressed the use of activating agents and secondary questions addressed assay temperature, length of analysis, pipetting, sample volume, reproducibility, and quality controls. Standardized, systematic evaluation of the literature was performed. Relevant articles were categorized according to level of evidence (LOE). Consensus was developed regarding conclusions for application of concepts to clinical practice. SETTING: Academic and referral veterinary medical centers. RESULTS: PubMed and CAB abstracts were searched. Twenty papers were initially identified concerning the primary question; 16 were in support of the questions (LOE 2 Good, LOE 3 Good, LOE 5 Good, LOE 6 Good, LOE 5 Fair, LOE 6 Fair); and 4 were neutral (LOE 3 Good, LOE 6 Good, LOE Fair, LOE 5 Fair). Additional papers were evaluated post hoc during manuscript preparation. CONCLUSIONS: Overall, there is a body of evidence from veterinary and human medicine that strongly suggests that TEG or ROTEM assays using citrated samples that employ an activator have significantly lower inherent variability than those that use recalcification alone. There is also strong evidence in dogs, cats, and humans that the results obtained using different activators are not directly comparable. There is no evidence to suggest that any one activating agent is superior to another for all patient populations, or drug monitoring indications. As such, use of more than one assay for complete thromboelastographic evaluation of a patient's coagulation system may be warranted. Standardization of the concentrations of activators would be beneficial.