Diagnosis of canine pulmonary thromboembolism by computed tomography and mathematical modelling using haemostatic and inflammatory variables.

There is no evidence-based diagnostic approach for diagnosis of pulmonary thromboembolism (PTE) in dogs. Many dogs with diseases that predispose to thrombosis are hypercoagulable when assessed with thromboelastography (TEG), but no direct link has been established. The aims of this study were: (1) to investigate if diseased dogs with PTE, diagnosed by computed tomography pulmonary angiography (CTPA), had evidence of hypercoagulability by TEG; (2) to characterise haemostatic and inflammatory changes in dogs with PTE; (3) to construct models for prediction of PTE based on combinations of haemostatic and inflammatory variables; and (4) to evaluate the performance of D-dimer measurement for prediction of PTE. Twenty-five dogs were included in this prospective observational study (PTE: n=6; non-PTE: n=19). Clot strength G values did not differ between the PTE and non-PTE groups in tissue factor (TF) or kaolin-activated TEG analyses. Haemostatic and inflammatory variables did not differ between the two groups. Linear discriminant analysis generated a model for prediction of PTE with a sensitivity and specificity of 100% when TF results were used as TEG data, and a model with sensitivity of 83% and specificity of 100% when kaolin results were used as TEG data. Receiver operating characteristic analysis of D-dimer levels showed that a value of >0.3mg/L yielded a sensitivity of 100% and a specificity of 71.4%. In conclusion, the study supports CTPA as method for diagnosing canine PTE, but shows that TEG alone cannot identify dogs with PTE. Models for prediction of PTE were generated, but require further validation.

Modulation of the activated protein C pathway in severe haemophilia A patients: The effects of thrombomodulin and a factor V-stabilizing fab.

INTRODUCTION: The thrombomodulin (TM)/activated protein C (APC) system is a key regulator of haemostasis, limiting amplification and propagation of the formed blood clot to the injury site. Dampening APC's inhibition of factor V (FV) and factor VIII (FVIII) may be a future strategy in developing next-generation therapeutic targets for haemophilia treatment. AIMS: To determine ex vivo the respective concentration-dependent effects of TM and a FV-stabilizing Fab on the APC regulatory pathway in severe FVIII-deficient blood and plasma. METHODS: Ten severe haemophilia A subjects and one healthy control were enrolled. Blood was spiked with TM (0, 1, 2.5, 5, 10, 20.0 nmol/L) and FV-stabilizing Fab (0, 3, 15, 65, 300 nmol/L). The respective effects were compared to FVIII concentrations of 3- and 10% using rotational thromboelastometry clotting time (CT) and thrombin generation analysis (TGA). RESULTS: With 1 and 2.5 nmol/L TM, 5% FVIII resulted in CT similar to the absence of TM, suggesting it completely reversed the effect of APC. Increasing TM concentrations also reduced peak thrombin generation and ETP. The addition of 300 nmol/L FV-stabilizing Fab returned CT to nearly baseline, but for most subjects was less than the effects of 3- or 10% FVIII. The FV-stabilizing Fab produced similar or greater thrombin generation compared to samples with 3- or 10% FVIII. CONCLUSIONS: The FV-stabilizing Fab resulted in enhanced CT and TGA parameters consistent with FVIII levels of 3- and 10%. Additional studies need to further characterize how modulating the APC pathway may prove beneficial in developing new haemophilia drug targets.

Texture analysis of pulmonary parenchymateous changes related to pulmonary thromboembolism in dogs - a novel approach using quantitative methods.

BACKGROUND: Diagnosis of pulmonary thromboembolism (PTE) in dogs relies on computed tomography pulmonary angiography (CTPA), but detailed interpretation of CTPA images is demanding for the radiologist and only large vessels may be evaluated. New approaches for better detection of smaller thrombi include dual energy computed tomography (DECT) as well as computer assisted diagnosis (CAD) techniques. The purpose of this study was to investigate the performance of quantitative texture analysis for detecting dogs with PTE using grey-level co-occurrence matrices (GLCM) and multivariate statistical classification analyses. CT images from healthy (n = 6) and diseased (n = 29) dogs with and without PTE confirmed on CTPA were segmented so that only tissue with CT numbers between -1024 and -250 Houndsfield Units (HU) was preserved. GLCM analysis and subsequent multivariate classification analyses were performed on texture parameters extracted from these images. RESULTS: Leave-one-dog-out cross validation and receiver operator characteristic (ROC) showed that the models generated from the texture analysis were able to predict healthy dogs with optimal levels of performance. Partial Least Square Discriminant Analysis (PLS-DA) obtained a sensitivity of 94% and a specificity of 96%, while Support Vector Machines (SVM) yielded a sensitivity of 99% and a specificity of 100%. The models, however, performed worse in classifying the type of disease in the diseased dog group: In diseased dogs with PTE sensitivities were 30% (PLS-DA) and 38% (SVM), and specificities were 80% (PLS-DA) and 89% (SVM). In diseased dogs without PTE the sensitivities of the models were 59% (PLS-DA) and 79% (SVM) and specificities were 79% (PLS-DA) and 82% (SVM). CONCLUSION: The results indicate that texture analysis of CTPA images using GLCM is an effective tool for distinguishing healthy from abnormal lung. Furthermore the texture of pulmonary parenchyma in dogs with PTE is altered, when compared to the texture of pulmonary parenchyma of healthy dogs. The models' poorer performance in classifying dogs within the diseased group, may be related to the low number of dogs compared to texture variables, a lack of balanced number of dogs within each group or a real lack of difference in the texture features among the diseased dogs.

Visualization of haemophilic arthropathy in F8-/- rats by ultrasonography and micro-computed tomography.

INTRODUCTION: A major complication of haemophilia is haemophilic arthropathy (HA), a debilitating disorder with an incompletely defined pathobiology. High-resolution imaging may provide new knowledge about onset and progression of HA, and thereby support identification of new treatment opportunities. Recently, a F8-/- rat model of HA was developed. The size of the rat allows for convenient and high resolution imaging of the joints, which could enable in vivo studies of HA development. AIM: To determine whether HA in the F8-/- rat can be visualized using ultrasonography (US) and micro-computed tomography (µCT). METHODS: Sixty F8-/- and 20 wild-type rats were subjected to a single or two induced knee bleeds. F8-/- rats were treated with either recombinant human FVIII (rhFVIII) or vehicle before the induction of knee bleeds. Haemophilic arthropathy was visualized using in vivo US and ex vivo µCT, and the observations correlated with histological evaluation. RESULTS: US and µCT detected pathologies in the knee related to HA. There was a strong correlation between disease severity determined by µCT and histopathology. rhFVIII treatment reduced the pathology identified with both imaging techniques. CONCLUSION: US and µCT are suitable imaging techniques for detection of blood-induced joint disease in F8-/- rats and may be used for longitudinal studies of disease progression.

Non-genetic risk factors in haemophilia A inhibitor management - the danger theory and the use of animal models.

In haemophilia A (HA) management, antidrug antibodies, or inhibitors, are a serious complication that renders factor VIII (FVIII) replacement therapy ineffective, increases morbidity and reduces quality of life for affected patients. Inhibitor development aetiology is multifactorial and covers both genetic and therapy related risk factors. Many therapy-related risk factors have proven difficult to confirm due to several confounding factors and the small study populations available. However, clinical studies indicate that e.g. on-demand treatment and surgery affect inhibitor development, and explanations for this association are being investigated. A potential explanation is the danger signal effect, where the immune response is activated by endogenous or exogenous danger or damage signals present at the time and site of FVIII administration. The danger theory explains how alarm signals from stressed, injured or dying cells can activate an immune reaction, without the involvement of foreign antigens. Bleeds, trauma, surgery or concomitant infection could be events initiating danger signalling in HA patients, resulting in an immune reaction towards administered FVIII that otherwise would pass unnoticed. This role of danger in HA inhibitor formation has previously been suggested, but a thorough discussion of this subject is lacking. The present review will discuss the potential role of danger signals in haemophilia and inhibitor development, with focus on treatment related risk factors with a suspected danger signal aetiology; on-demand treatment, treatment during major bleeds or surgery, and treatment during infection or vaccination. Clinical studies as well as animal experiments addressing these factors will be reviewed.

Joint bleeds increase the inhibitor response to human factor VIII in a rat model of severe haemophilia A.

INTRODUCTION: The most serious complication in haemophilia A (HA) replacement therapy with coagulation factor VIII (FVIII) is neutralizing antibodies, i.e. inhibitors. It has been hypothesized that danger signals generated during a bleed might have an adjuvant effect on the immune response to FVIII in on-demand treatment, increasing the inhibitor risk. AIM: To compare the antibody response to treatment with recombinant human FVIII (rhFVIII) in relation to induced knee joint bleeds and treatment without concurrent bleeds in a HA rat model. METHOD: HA rats were divided into two groups: one group (n = 10) receiving three needle induced knee joint bleeds 14 days apart and a control group (n = 9) receiving three sham procedures. Three hours after each injury/sham 50 IU kg(-1) rhFVIII was administrated intravenously. Subsequently, both groups continued rhFVIII treatment for another 9 weeks. Binding antibodies were analysed using an enzyme-linked immunosorbent assay and neutralizing antibodies using a Bethesda-like assay. RESULTS: Rats in the knee-bleed group developed a significantly faster inhibitor response and reached significantly higher inhibitor levels. In the knee-bleed group, 80% developed inhibitors vs. 33% in the control group, demonstrating a 2.4 times higher inhibitor risk when treating concurrent with bleeds. CONCLUSION: FVIII treatment in relation to a bleed potentiates inhibitor development compared to FVIII treatment alone in this HA rat, indicating that bleeding is a potential danger signal. Our results support the theory that FVIII replacement therapy concurrent with a bleeding episode increases the inhibitor risk, which to the best of our knowledge, has not been confirmed in an animal model before.

The F8(-/-) rat as a model of hemophilic arthropathy.

UNLABELLED: Essentials Validating the F8 rat as a new intermediate-size animal model of hemophilic arthropathy. Factor VIII (FVIII) treated F8(-/-) rats suffered induced hemarthrosis analyzed by histopathology. F8 (-/-) animals develop hemophilic arthropathy upon hemarthrosis, preventable by FVIII treatment. The F8 (-/-) rat presents as a new pharmacologic model of hemophilic arthropathy. SUMMARY: Background Translational animal models of hemophilia are valuable for determining the pathobiology of the disease and its co-morbidities (e.g. hemophilic arthropathy, HA). The biologic mechanisms behind the development of HA, a painful and debilitating condition, are not completely understood. We recently characterized a F8(-/-) rat, which could be a new preclinical model of HA. Objectives To establish the F8(-/-) rat as a model of HA by determining if the F8(-/-) rat develops HA resembling human HA after an induced joint bleed and whether a second joint bleed causes further disease progression. Methods Wild-type and F8(-/-) rats were treated with vehicle or recombinant human factor VIII (rhFVIII) prior to a needle-induced joint bleed. Joint swelling was measured prior to injury, the following 7 days and upon euthanasia. Histologic sections of the joint were stained, and athropathic changes identified and scored with regard to synovitis, bone remodelling, cartilage degradation and hemosiderin deposition. Results Vehicle-treated F8(-/-) rats experienced marked joint swelling and developed chronic degenerative joint changes (i.e. fibrosis of the subsynovial membrane, chondrocyte loss and excessive bone remodeling). Treatment with rhFVIII reduced or prevented swelling and degenerative joint changes, returning the F8(-/-) animals to a wild-type phenotype. Conclusion The hemophilic phenotype of the F8(-/-) rat resulted in a persistent hemarthrosis following an induced joint bleed. This caused development of HA resembling human HA, which was prevented by rhFVIII treatment, confirming the potential of the F8(-/-) rat as a model of HA.

Antibody response to recombinant human coagulation factor VIII in a new rat model of severe hemophilia A.

BACKGROUND: Neutralizing antibodies toward FVIII replacement therapy (inhibitors) are the most serious treatment-related complication in hemophilia A (HA). A rat model of severe HA (F8(-/-) ) has recently been developed, but an immunological characterization is needed to determine the value of using the model for research into inhibitor development. OBJECTIVES: Characterize the antibody response towards recombinant human coagulation factor VIII (rhFVIII) in the HA rat, following a human prophylactic dosing regimen. METHODS: Two identical studies were performed, which included a total of 17 homozygous HA rats (F8(-/-) , 0% FVIII activity), 12 heterozygous rats (F8(+/-) ), and 12 wild-type (F8(+/+) ) rats. All rats received intravenous injections of rhFVIII at 50 IU kg(-1) twice weekly for 4 weeks. Predosing blood samples were analyzed for binding and neutralizing anti-rhFVIII antibodies at weeks 1-7. RESULTS: In both studies, antibodies developed after 4-6 administrations of rhFVIII, and neutralizing antibodies reached levels similar to human patients (range 1-111 BU, median 6.0 BU) at the end of the study. There was no significant difference between the two studies or between genotypes in time to response or levels reached for binding and neutralizing antibodies. Interestingly, early spontaneous bleeds were associated with a faster antibody response. CONCLUSIONS: Following intravenous administration of human FVIII, according to a clinical prophylaxis regimen, a robust and reproducible antibody response is seen in this HA rat model, suggesting that the model is useful for intervention studies with the aim of suppressing, delaying, or preventing the inhibitor response. Also, bleeds seem to have an adjuvant effect on the immune response.

A novel F8 -/- rat as a translational model of human hemophilia A.

BACKGROUND: In preclinical hemophilia research, an animal model that reflects both the phenotype and the pathology of the disease is needed. OBJECTIVES: Here, we describe the generation and characterization of a novel genetically engineered F8(-/-) rat model. METHODS: The rats were produced on a Sprague Dawley background with the zinc finger nuclease technique. A founder with a 13-bp deletion in exon 16 causing a premature translational stop in the C-terminal part of the A3 domain of factor VIII was selected, and a breeding colony was established. RESULTS: Seventy per cent of the homozygous rats had clinically manifest spontaneous hemorrhagic episodes that needed treatment. The F8(-/-) rats had no detectable FVIII activity, and had a significantly prolonged activated partial thromboplastin time (APTT) and clot formation time as compared with wild-type (WT)/WT rats. In vitro spiking of rat plasma with human recombinant FVIII resulted in dose-dependent normalization of the APTT. CONCLUSION: On the basis of the targeted deletion in F8, and the distinct physical and analytic characteristics of the rat, we conclude that an FVIII-deficient rat strain has been generated that has the potential to contribute greatly to translational research.

Disseminated intravascular coagulation in a novel porcine model of severe Staphylococcus aureus sepsis fulfills human clinical criteria.

Sepsis is a common and often fatal complication in human patients in intensive care units. Relevant and well characterized animal models of sepsis may provide valuable information on pathophysiological mechanisms and be a mean of testing new therapeutic strategies. Large animal models of Staphylococcus aureus sepsis are rare, even though S. aureus increasingly affects human patients. Sepsis changes the haemostatic balance and leads to endothelial cell (EC) activation, coagulopathy and, in severe cases, disseminated intravascular coagulation (DIC). The aim of this study was to characterize the haemostatic and vascular alterations in a novel porcine model of severe S. aureus sepsis, investigating whether the changes fulfill the human clinical criteria for DIC. Five pigs were inoculated intravenously with S. aureus and two control animals were sham-inoculated. Blood samples were collected for thromboelastography (TEG) and assessment of plasma-based haemostatic parameters. Tissue was collected for histopathology and reverse transcriptase quantitative real-time polymerase chain reaction for measurement of mRNA encoding EC markers. All infected animals developed DIC; including procoagulant activation represented by hypercoagulable TEG profiles and prolonged clotting time. Histologically, numerous pulmonary thrombi were present in one pig. Inhibitor consumption was represented by decreasing antithrombin levels in infected pigs. Hyaline globules were found in three infected pigs, confirming fibrinolytic activation. EC activation was identified by expression of von Willebrand factor in small vessels together with elevated mRNA encoding activated EC markers. Severe haemostatic and vascular changes fulfilling the human criteria for DIC were therefore seen in all infected pigs. A tendency towards uncompensated DIC was seen in two animals.

Hemostatic abnormalities in uncomplicated babesiosis (Babesia rossi) in dogs.

BACKGROUND: Babesiosis in dogs is associated with severe thrombocytopenia; yet infected dogs rarely show clinical signs of hemorrhage. HYPOTHESIS: Dogs with uncomplicated babesiosis have normal hemostatic capacity despite severe thrombocytopenia. ANIMALS: Nineteen client-owned dogs with uncomplicated babesiosis; 10 healthy controls. METHODS: A prospective, cross-sectional, observational study. Thromboelastography (TEG), prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, D-dimers, and antithrombin (AT) measured in both groups. Statistical significance set at P < .01. RESULTS: Babesiosis group hematocrit and platelet count significantly lower than controls (0.29 versus 0.50 L/L; P < .001 and 20.0 versus 374.5 × 10(9)/L; P < .001, respectively). Except for K, no significant difference in TEG variables between groups. Hemostatic variables for babesiosis group versus controls (mean ± SD); R: 5.9 ± 1.8 versus 4.6 ± 0.9 min (P = .048); K: 2.8 ± 1.1 versus 1.9 ± 0.6 min (P = .003); angle: 55.5 ± 11.7 versus 62.2 ± 4.1 degrees (P = .036); MA: 48.4 ± 9.7 versus 57.2 ± 5.2 mm (P = 0.013); G: 5.1 ± 1.9 versus 6.9 ± 1.5 dyn/cm(2) (P = .019); LY30 (median, range): 0 (0-5.7) versus 0.6% (0-6.1) (P = .152); and LY60: 0 (0-8.8) versus 3.1% (0-13.1) (P = .012). AT activity significantly lower (105.2 ± 16.5 versus 127.8 ± 15.4%; P = .001). Fibrinogen concentration significantly higher in babesiosis group (5.7 ± 1.3 versus. 3.0 ± 0.7 g/L; P < .001). CONCLUSION AND CLINICAL IMPORTANCE: Despite severe thrombocytopenia, dogs with uncomplicated babesiosis did not have clinical signs of hemorrhage and TEG variables were normal, which could indicate a normocoagulable state.

The impact of Staphylococcus aureus concentration on the development of pulmonary lesions and cytokine expression after intravenous inoculation of pigs.

Acute respiratory distress syndrome is a common complication in severe sepsis. In pigs, the lungs play an important role in clearing systemic bacterial infections due to pulmonary intravascular macrophages found specifically in pigs. However, this increases the exposure of the porcine lungs to pathogens and potential injury. The authors propose that increasing the concentration of the inoculum without changing the bacterial dose will lead to severe sepsis with pronounced pulmonary lesions. This could potentially create a risk of cytokine spillover to the circulation, leading to an increased systemic response. Eight Danish Landrace pigs, approximately 10 weeks old, were inoculated twice with a low or once with a high concentration of Staphylococcus aureus. Three pigs were sham-inoculated. The animals were grouped based on macro- and microscopic lung lesions. The mRNA expression of local pulmonary inflammatory markers was compared to protein levels of systemic inflammatory markers. The most severe pulmonary lesions were observed in animals receiving the high S. aureus concentration, indicating that severity of lesions is dependent on inoculum concentration rather than total numbers of bacteria. Furthermore, local mRNA expression of inflammatory cytokines appeared to be dependent on the magnitude and severity of tissue destruction, including the ability to confine the lesions. Increasing mRNA levels of serum amyloid A could be a confident marker of severity of pulmonary lesions. Since no correlation was observed between local and systemic levels of inflammatory cytokines, this finding could indicate an ability of the porcine lung to compartmentalize the local inflammatory response and thus restrict systemic contribution.

Serial assessment of the coagulation status of dogs with immune-mediated haemolytic anaemia using thromboelastography.

This study investigated the coagulation status of dogs with immune-mediated haemolytic anaemia (IMHA) over time. Thirty animals with primary IMHA were blood sampled on three occasions over a 5 day period and assays performed included prothrombin time, activated partial thromboplastin time, D-dimer and fibrinogen concentration, antithrombin activity and recalcified unactivated thromboelastography (TEG). Based on TEG, dogs with IMHA were significantly hypercoagulable vs. controls (P<0.001) and over the 5 day period, 3/4 of the TEG parameters reflected increased clotting kinetics (P ≤ 0.02). The 30 day survival of these patients was 80% and, at hospital admission, the TEG maximum amplitude (MA) was significantly higher in survivors than non-survivors (P=0.015). Each unit increase in MA was associated with an increased odds of 30 day survival of 1.13 (95%; CI 1.02-1.25). Based on TEG, most dogs with IMHA were hypercoagulable on admission and their clotting kinetics increased with time. Relative hypocoagulability identified by TEG at initial assessment was found to be a negative prognostic indicator.

Obesity increases initial rate of fibrin formation during blood coagulation in domestic shorthaired cats.

Obesity predisposes to a prothrombotic state in humans, but whether a similar state occurs in obese animals is unknown. The objective of the current study was to examine the effect of body fat percentage (BF) on haemostatic parameters including thromboelastography with tissue factor as activator (TF-TEG) in client owned indoor-confined physically inactive cats. Seventy-two cats were included following an initial thorough health examination, and a complete blood count, biochemistry panel, conventional coagulation panel and a TF-TEG analysis were performed with tissue factor (1:50,000) as activator. The cats were anaesthetized, and BF was measured using Dual-energy X-ray absorptiometry. Significant difference between lean (BF < 35%, n = 26), overweight (35% < BF < 45%, n = 28) and obese (BF > 45%, n = 18) cats was identified using ANOVA. The correlation between BF, serum leptin and total adiponectin, respectively, with individual TEG and conventional coagulation parameters was evaluated. Obese cats showed a faster rate of fibrin formation (TF-TEG(R), p < 0.05), and TF-TEG(R) was positively correlated with plasma leptin levels. Increasing BF did not affect other conventional coagulation or TF-TEG parameters. In conclusion, this study indicates a connection between body fat content and altered haemostasis, also in cats. Whether feline obesity causes a hypercoagulable state of clinical relevance should be further investigated.

The presence of antiphospholipid antibodies in healthy Bernese Mountain Dogs.

BACKGROUND: The role of antiphospholipid antibodies in the prolonged activated partial thromboplastin time (aPTT) previously identified in healthy Bernese Mountain Dogs remains unknown. In people, an isolated prolonged aPTT without evidence of bleeding might be because of a thrombophilic condition caused by antiphospholipid antibodies. OBJECTIVE: To examine if prolonged aPTT in healthy Bernese Mountain Dogs is because of antiphospholipid antibodies. ANIMALS: Twenty-two healthy Bernese Mountain Dogs and 10 healthy adult dogs of various breeds. METHODS: Prospective case control study. Healthy Bernese Moutain Dogs were examined twice over 6 months. Dogs were investigated for the presence of lupus anticoagulants and anticardiolipin (aCL) antibodies by the use of multiple aPTT tests with low and high lupus anticoagulant sensitivities, a mixing study, and an ELISA test for aCL antibody optical density to detect solid phase antiphospholipid antibodies. RESULTS: In all, 15 of 22 healthy Bernese Mountain Dogs were positive for lupus anticoagulants. The Bernese Mountain Dogs had markedly higher levels of aCL antibodies compared with the control dogs (P = .006). In all, 7 of 21 of the Bernese Mountain Dogs were positive for both lupus anticoagulants and aCL antibodies, whereas 4 of 21 Bernese Mountain Dogs were negative for both. CONCLUSIONS AND CLINICAL IMPORTANCE: Lupus anticoagulants and aCL antibodies could be the cause of prolonged aPTT in healthy Bernese Mountain Dogs. The importance of the antiphospholipid antibodies in the dogs remains unknown.

Use of serum concentrations of interleukin-18 and monocyte chemoattractant protein-1 as prognostic indicators in primary immune-mediated hemolytic anemia in dogs.

BACKGROUND: The cytokine response in immune-mediated hemolytic anemia (IMHA) is poorly characterized and correlation with outcome is unknown. HYPOTHESIS/OBJECTIVES: To determine if cytokine activity is correlated with outcome in dogs with IMHA. ANIMALS: Twenty dogs with primary IMHA and 6 control dogs. METHODS: Prospective study on dogs with IMHA with blood sampling at admission. Serum activity of interleukin-2 (IL-2), IL-4, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-inducible protein-10, interferon-gamma, and keratinocyte chemoattractant (KC) was assessed. RESULTS: Thirty-day case fatality rate was 25% (5/20 dogs). Increased concentrations (median [range]) of IL-2 (45.5 ng/L [0;830] versus 0 ng/L [0;46.8]), IL-10 (8.2 ng/L [0;60.6] versus 0 ng/L [0;88.2]), KC (1.7 µg/L [0.3;4.7] versus 0.5 µg/L [0.2;1.1]), and MCP-1 (162 ng/L [97.6;438] versus 124 ng/L [90.2;168]) were observed in dogs with IMHA compared with controls. The cytokine profile was indicative of a mixture of pro- and anti-inflammatory cytokines of various cellular origins. Cytokines/chemokines strongly associated with macrophage/monocyte activation and recruitment were significantly increased in nonsurvivors compared with survivors; IL-15 (179 ng/L [48.0;570] versus 21.3 ng/L [0;193]), IL-18 (199 ng/L [58.7;915] versus 37.4 ng/L [0;128]), GM-CSF (134 ng/L [70.0;863] versus 57.6 ng/L [0;164]), and MCP-1 (219 ng/L [135;438] versus 159 ng/L [97.6;274]), respectively. Logistic regression suggested increased IL-18 and MCP-1 concentrations were independently associated with mortality in this population (P<.05, Wald's type 3). CONCLUSIONS AND CLINICAL IMPORTANCE: A mixed cytokine response is present in dogs with IMHA and mediators of macrophage activation and recruitment might serve as prognostic indicators.

Thromboelastographic evaluation of hemostatic function in dogs with disseminated intravascular coagulation.

BACKGROUND: There is considerable variation in the coagulation profile of dogs with disseminated intravascular coagulation (DIC), making it difficult to assess overall hemostatic function. OBJECTIVES: To characterize the overall hemostatic state in dogs with DIC, by use of tissue factor-activated thromboelastography (TF-TEG), and to determine whether there is an association between hemostasis and outcome. ANIMALS: 50 dogs with DIC. METHODS: Dogs admitted to the intensive care units, with an underlying disease known to predispose to DIC, were prospectively assessed with TF-TEG. Citrated blood samples were collected daily during hospitalization and an extended coagulation panel and TF-TEG were performed. Diagnosis of DIC was based on expert opinion. RESULTS: Hemostatic dysfunction was observed on the TF-TEG profile in 33/50 of the dogs, of which 22/50 were hypercoagulable and 11/50 were hypocoagulable based on the TF-TEG G value alone. There were significant differences in k, alpha, and MA values (P < .0001) among hypo-, normo-, and hypercoagulable dogs. There was a significant difference in case fatality rate between hypo- (64%) and hypercoagulable (32%) dogs (relative risk = 2.38; P= .04). Dogs that died had significantly lower antithrombin activity (P= .03) and higher d-dimer concentration (P= .03) than survivors. CONCLUSIONS: The most common overall hemostatic abnormality in dogs diagnosed with DIC was hypercoagulability, and there was significant difference in survival between hyper- and hypocoagulable dogs. The results suggest TF-TEG is valuable in the assessment of hemostatic function in dogs diagnosed with DIC.

Evaluation of human recombinant tissue factor-activated thromboelastography in 49 dogs with neoplasia.

BACKGROUND: Abnormal routine coagulation assay results have been reported to be common in veterinary patients with neoplasia, but the overall hemostatic functional state, including hypercoagulability, has not been described. HYPOTHESIS: The overall hemostatic functional state, including hypercoagulability, can be assessed in dogs with neoplasia by tissue factor (TF)-activated thromboelastography (TEG). ANIMALS: Thirty-six dogs with malignant neoplasia and 13 dogs with benign neoplasia presented to the Small Animal Veterinary Teaching Hospital, The University of Copenhagen, Frederiksberg, Denmark. METHODS: Prospective study evaluating the overall hemostatic functional state in dogs with neoplasia by a newly validated TF-activated TEG assay and routine coagulation parameters activated partial thromboplastin time (aPTT), prothrombin time (PT), platelet count, and D-dimer concentration. RESULTS: Hemostatic dysfunction was observed in 28/49 (57%) dogs with neoplasia. Twenty-four were dogs with malignant neoplasia, the majority of which 18/36 (50%) were hypercoagulable, whereas 6/36 (17%) were hypocoagulable. All hypocoagulable dogs had metastatic disease. The proportion of dogs with altered hemostasis was significantly different between dogs with malignant and benign neoplasia. CONCLUSIONS AND CLINICAL IMPORTANCE: TF-activated TEG detected hypercoagulable and hypocoagulable states in this population of dogs with neoplasia. The most common hemostatic abnormality in dogs with malignant neoplasia was hypercoagulability. These findings suggest that this novel hemostatic function test may be of value as a cage side method for the assessment of overall hemostatic function in dogs with cancer, including the detection of both hyper- and hypocoagulable states as well as mixed disorders.

Cellular, hemostatic, and inflammatory parameters of the surgical stress response in pigs undergoing partial pericardectomy via open thoracotomy or thoracoscopy.

BACKGROUND: Thoracoscopy has been shown to reduce the inflammatory and immunologic response to surgical stress, as compared with corresponding open procedures in humans. The influence on the hemostatic system, however, has not been thoroughly evaluated. The current study aimed to compare the perioperative and immediate postoperative changes in cellular, hemostatic, and inflammatory parameters after a partial pericardectomy performed by either thoracoscopy or thoracotomy. METHODS: For this study, 16 pigs were randomly assigned to have a partial pericardectomy performed thoracoscopically or by thoracotomy. Blood was collected intraoperatively, then 10 min, 3 h, and 6 h after surgery. Whole ethylenediaminetetraacetic acid (EDTA)-stabilized blood and plasma were examined for cellular, hemostatic, and inflammatory parameters, respectively, and thromboelastography (TEG) was performed on citrated whole blood. RESULTS: No significant difference in any of the parameters measured was found between the two groups except for the TEG parameter R-time, which was significantly shorter in the thoracoscopic group 3 h postoperatively. In both groups, a significant postoperative state of hypercoagulability and increase in inflammatory parameters was found. Additionally, pig blood showed a high degree of hypercoagulability in preoperative measurements, as compared with other species. CONCLUSIONS: Partial pericardectomy performed by thoracotomy or thoracoscopy in pigs produces a surgical stress response of equal magnitude, as measured by cellular, hemostatic, and inflammatory changes.