Clinical bleeding diathesis, laboratory haemostatic aberrations and survival in dogs infected with Angiostrongylus vasorum: 180 cases (2005-2019).

OBJECTIVES: Bleeding diathesis is a complication in dogs infected with Angiostrongylus vasorum. This retrospective study investigated clinical and laboratory haemostatic differences in A. vasorum-positive dogs with and without signs of bleeding and impact of bleeding on survival. MATERIALS AND METHODS: Demographics, type of clinical bleeding, haematocrit and a range of haemostatic tests, including thromboelastography and derived velocity curves were retrospectively registered from A. vasorum-positive dogs. All parameters were compared between dogs with and without signs of bleeding using univariable analyses. Binomial and multinomial regression models were applied to examine specific indicators in the bleeding dogs. P-values were false discovery rate adjusted, and adjusted P<0.05 was considered significant. RESULTS: One hundred and eighty dogs entered the study, including 65 dogs (36.1%) presenting with bleeding diathesis. Different types of cutaneous and mucosal bleeding were the most common clinical findings. Twenty dogs presented with neurological signs associated with intracranial and intra-spinal bleeding. One hundred and thirty-seven dogs had haematological and/or haemostatic laboratory analyses performed. Haematocrit, platelet count, thromboelastographic angle, maximum amplitude, global clot strength, maximum rate of thrombin generation and total thrombin generation were decreased, while prothrombin time was prolonged in bleeding dogs. Survival rate of bleeding dogs was lower at hospital discharge (76.9%) and 1 month after diagnosis (66.0%) than in dogs without signs of bleeding (94.8% and 90.1% at discharge and at 1 month, respectively). CLINICAL SIGNIFICANCE: Several haemostatic aberrations were detected in A. vasorum-positive dogs with bleeding diathesis. Bleeding was identified as an important negative prognostic indicator in A. vasorum-positive dogs.

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.

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.

Pharmacokinetics, pharmacodynamics and safety of recombinant canine FVIIa in a study dosing one haemophilia A and one haemostatically normal dog.

Recombinant human FVIIa (rhFVIIa) corrects the coagulopathy in hemophilia A and B as well as FVII deficiency. This is also the case in dogs until canine anti-human FVIIa antibodies develop (~2 weeks). Recombinant canine factor VIIa (rcFVIIa), successfully over-expressed by gene transfer in haemophilia dogs, has provided long-term haemostasis (>2 years). However, pharmacokinetics (PK), pharmacodynamics (PD) and safety of rcFVIIa after pharmacological administration have not been reported. We therefore wanted to explore the safety, PK and PD of rcFVIIa in dogs. A pilot study was set up to evaluate the safety as well as PK and PD of rcFVIIa after a single intravenous dose of 270 µg kg(-1) to one HA and one haemostatically normal dog and to directly compare rcFVIIa with rhFVIIa in these two dogs. Single doses of rcFVIIa and rhFVIIa were well tolerated. No adverse events were observed. Pharmacokinetic characteristics including half-life (FVIIa activity: 1.2-1.8 h; FVIIa antigen 2.8-3.7 h) and clearance were comparable for rcFVIIa and rhFVIIa. Kaolin-activated thromboelastography approached normal in the HA dog with the improvement being most pronounced after rcFVIIa. This study provided the first evidence that administering rcFVIIa intravenously is feasible, safe, well tolerated and efficacious in correcting the haemophilic coagulopathy in canine HA and that rcFVIIa exhibits pharmacokinetic characteristics comparable to rhFVIIa in haemophilic and haemostatically competent dogs. This strengthens the hypothesis that rcFVIIa can be administered to dogs to mimic the administration of rhFVIIa to humans.

Retrospective review of 27 European cases of fatal elephant endotheliotropic herpesvirus-haemorrhagic disease reveals evidence of disseminated intravascular coagulation.

Elephant endotheliotropic herpesvirus haemorrhagic disease (EEHV-HD) is widely acknowledged as the most common cause of mortality in young Asian elephants (Elephas maximus) in captivity. The objective of the current study was to perform a blinded, retrospective pathology review of European EEHV-HD fatalities, constituting the largest systematic assessment of EEHV-HD pathology to date. Findings between viral genotypes were compared with the aim to investigate if disseminated intravascular coagulation (DIC) could be substantiated as a significant complicating factor, thereby increasing the understanding of disease pathophysiology. Immunohistochemical staining confirmed endothelial cell (EC) damage and the presence of EC intranuclear inclusion bodies, demonstrating a direct viral cytopathic effect. Microthrombi were observed in 63% of cases in several organs, including lungs, which, together with widespread haemorrhage and thrombocytopenia reported in EEHV-HD case reports, supports the presence of overt DIC as a serious haemostatic complication of active EEHV infection. Death was attributed to widespread vascular damage with multi-organ dysfunction, including severe acute myocardial haemorrhage and subsequent cardiac failure. Systemic inflammation observed in the absence of bacterial infection may be caused by cytokine release syndrome. Findings reinforce the necessity to investigate cytokine responses and haemostatic status during symptomatic and asymptomatic EEHV viraemia, to potentially support the use of anti-inflammatory treatment in conjunction with anti-viral therapy and cardiovascular support.

IL-1 beta, IL-6, KC and MCP-1 are elevated in synovial fluid from haemophilic mice with experimentally induced haemarthrosis.

The hallmark of haemophilia is the joint morbidity resulting from haemarthrosis that accounts for the majority of the bleeds. The exact mechanisms underlying changes are not fully elucidated. Cytokines are speculated to be involved in the progression and in vitro studies have confirmed the presence of elevated levels of cytokines in synovial tissue and cartilage from patients with haemophilic synovitis. In this study, the presence of selected cytokines in synovial fluid from haemophilia A mice with experimentally induced haemarthroses treated with rFVIII, rFVIIa and an rFVIIa analogue were investigated. Ten cytokines previously shown to be involved in arthritic syndromes were evaluated. Interleukin (IL)-1 beta, IL-2, IL-4, IL-6, IL-10, IL-17, Tumor Necrosis Factor-alpha (TNF- alpha), keratinocyte-derived chemokine (KC), Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES) and monocyte chemotactic protein-1 (MCP-1) were included. In this article, we demonstrate, for the first time, that bleeding in knee joints of haemophilia A mice resulted in correlated increased levels of the pro-inflammatory cytokines: IL-1 beta, IL-6, KC and the MCP-1 in synovial fluid. These results suggest an important role of MCP-1 in the recruitment of monocytes and furthermore that the inflamed synovium releases IL-1 beta, IL-6 and KC, which in turn might contribute to further progression of the inflammatory process.

Changes in platelet function in Dachshunds with early stages of myxomatous mitral valve disease.

The aim of this study was to evaluate platelet function in Dachshunds during early stages of myxomatous mitral valve disease. Clinical examination and echocardiography were performed in 34 wirehaired standard sized Dachshunds. Platelet function was evaluated using the PFA-100 (reported as closure time). In addition, whole blood platelet aggregation response and hemostatic markers were evaluated. Significant longer PFA-100 closure time (CT) was found in 12 Dachshunds with mild mitral regurgitation (MR) compared to 22 Dachshunds with minimal MR. Only five Dachshunds responded to adenosine diphosphate in the whole blood aggregation analyses. There were no differences between the two dog groups in plasma fibrinogen, plasma von Willebrand factor (vWf) or vWf multimer distribution; however, there was a significant correlation between CT and plasma vWf concentration and CT and plasma fibrinogen concentration. The higher CT found in Dachshunds with mild MR suggests a form of platelet dysfunction in Dachshunds with MR.

In vivo models of haemophilia - status on current knowledge of clinical phenotypes and therapeutic interventions.

Animal models have contributed immensely to the understanding of and the improvement in treatment of haemophilia A and B. First, establishment of haemophilic dog colonies provided an invaluable opportunity to investigate the diseases and later, the advances in gene technologies resulting in small haemophilic animal models were a milestone in the preclinical research making it possible to address some of the many unanswered questions. This review provides an overview of animal models used in the study of haemophilia as well as a short overview of the contributions resulting from studies in these models.

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.

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.

Liposome-encapsulated chemotherapy: Current evidence for its use in companion animals.

Cytotoxic drugs encapsulated into liposomes were originally designed to increase the anticancer response, while minimizing off-target adverse effects. The first liposomal chemotherapeutic drug was approved for use in humans more than 20 years ago, and the first publication regarding its use in a canine cancer patient was published shortly thereafter. Regardless, no general application for liposomal cytotoxic drugs has been established in veterinary oncology till now. Due to the popularity of canines as experimental models for pharmacokinetic analyses and toxicity studies, multiple publications exist describing various liposomal drugs in healthy dogs. Also, some evidence for its use in veterinary cancer patients exists, especially in canine lymphoma, canine splenic hemangiosarcoma and feline soft tissue sarcoma, however, the results have not been overwhelming. Reasons for this may be related to inherent issues with the enhanced permeability and retention effect, the tumour phenomenon which liposomal drugs exploit. This effect seems very heterogeneously distributed in the tumour. Also, it is potentially not as ubiquitously occurring as once thought, and it may prove important to select patients for liposomal therapy on an individual, non-histology-oriented, basis. Concurrently, new developments with active-release modified liposomes in experimental models and humans will likely be relevant for veterinary patients as well, and holds the potential to improve the therapeutic response. It, however, does not resolve the other challenges that liposomal chemotherapy faces, and more work still needs to be done to determine which veterinary patients may benefit the most from liposomal chemotherapy.

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.

Efficacy and safety of imidacloprid/moxidectin spot-on solution and fenbendazole in the treatment of dogs naturally infected with Angiostrongylus vasorum (Baillet, 1866).

A randomized, blinded, controlled multicentre field trial study was conducted to evaluate the efficacy and safety of imidacloprid 10%/moxidectin 2.5% spot-on solution and fenbendazole in treating dogs naturally infected with Angiostrongylus vasorum. Dogs were randomly treated either with a single dose of 0.1 ml/kg bodyweight of imidacloprid 10%/moxidectin 2.5% spot-on solution or with 25 mg/kg bodyweight fenbendazole per os for 20 days. The study period was 42 days with dogs being examined on days 0, 7 and 42. The primary efficacy parameter was the presence of L1 larvae in faecal samples evaluated by a Baermann test from three consecutive days. Thoracic radiographs performed on each visit were being taken as a paraclinical parameter to support the results of the Baermann test. Twenty-seven dogs in the imidacloprid/moxidectin group and 23 dogs in the fenbendazole group completed the study according to protocol. The efficacies of the two treatment protocols were 85.2% (imidacloprid/moxidectin) and 91.3% (fenbendazole) with no significant difference between treatment groups. On radiographic evaluation pulmonary parenchyma showed similar improvement in each group. No serious adverse effects to treatment were recorded: most of the minor adverse effects were gastrointestinal such as diarrhea (nine dogs), vomitus (eight dogs) and salivation (three dogs). In general, these adverse effects were of short duration (1-2 days) within the first few days after treatment start and required little or no treatment. This prospective study demonstrates that both treatment protocols used are efficacious under field conditions, that treatment of mildly to moderately infected dogs with either of these protocols is safe and yields an excellent prognosis for recovering from the infection.

Effects of breed, gender, exercise and white-coat effect on markers of endothelial function in dogs.

This study examines how systemic biomarkers of endothelial function and nitric oxide metabolism are affected by exercise in dogs. Furthermore, breed variation and white-coat effect have been tested by sampling three different dog breeds both in their home and in a clinical setting. Short-term exercise increased plasma nitrate and nitrite (NOx) and von Willebrand factor (vWf). There was significant difference between Pointers and the small dog breeds Cairn Terriers and Cavalier King Charles Spaniels in the general plasma levels of vWf and asymmetric dimethylarginine (ADMA). NOx and vWf were significantly higher when the sample was taken in the laboratory cf. at home, whereas ADMA and L-arginine were significantly lower. In conclusion, both short-term exercise and white-coat effect influence several plasma markers of endothelial function depending also on the breed and gender of the dogs. These findings should be considered in future studies concerning endothelial function in dogs.

Circulating let-7g is down-regulated in Bernese Mountain dogs with disseminated histiocytic sarcoma and carcinomas - a prospective study.

Cancer is a prevalent cause of mortality in Bernese mountain dogs (BMDs). Circulating microRNAs (miRNAs) are found in blood and have been identified as promising biomarkers in various neoplastic diseases in humans. In the current study, the expression profile of different types of miRNAs was investigated in healthy BMDs and BMDs with cancer. Seven healthy and six non-treated BMDs with cancer [four with disseminated histiocytic sarcomas (DHS)] were enrolled in this study. Clinical evaluations including physical examination, blood analysis, urinalysis and diagnostic imaging were performed on all dogs. Twenty-four different miRNAs were profiled from RNA isolated from whole blood preserved in PAXgene® tubes using quantitative real-time PCR (qPCR). The miRNA let-7g was significantly down-regulated in dogs with cancer (P = 0.002) and dogs with DHS (P = 0.011) compared with healthy controls. This miRNA is a known tumour suppressor and further analyses are warranted to assess its value as a non-invasive biomarker for early detection of different types of cancer in BMDs.

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.

Expression of tissue factor in canine mammary tumours and correlation with grade, stage and markers of haemostasis and inflammation.

Tissue factor (TF) expression in human cancers has been associated with a procoagulant state and facilitation of metastasis. This study was conducted in order to evaluate if TF was expressed in canine mammary tumours. Forty epithelial mammary tumours from 28 dogs were included. TF expression of the tumours was evaluated by immunohistochemistry using a polyclonal antibody against recombinant canine TF. In addition, thromboelastography, haemostatic and inflammatory parameters were evaluated in the patients. TF was recognized in 44% of benign and 58% of malignant tumours. TF localized to the cytoplasmic membrane of neoplastic luminal epithelial cells and/or diffusely in the cytoplasm. No association was found between TF expression and stage or grade of disease. A significant association between TF expression and antithrombin and plasminogen was found, and extensive TF expression was seen in a lymph node metastasis classified as anaplastic mammary carcinoma from a dog with concomitant disseminated intravascular coagulation (DIC).

Novel, high incidence exercise-induced muscle bleeding model in hemophilia B mice: rationale, development and prophylactic intervention.

INTRODUCTION: Muscle hematomas are the second most common complication of hemophilia and insufficient treatment may result in serious and even life-threatening complications. Hemophilic dogs and rats do experience spontaneous muscle bleeding, but currently, no experimental animal model is available specifically investigating spontaneous muscle bleeds in a hemophilic setting. AIM: The objective of this study was to develop a model of spontaneous muscle bleeds in hemophilia B mice. We hypothesized that treadmill exercise would induce muscle bleeds in hemophilia B mice but not in normal non-hemophilic mice and that treatment with recombinant factor IX (rFIX) before treadmill exercise could prevent the occurrence of pathology. METHODS: A total of 203 mice (123 F9-KO and 80 C57BL/6NTac) were included in three separate studies: (i) the model implementation study investigating the bleeding pattern in hemophilia B mice after treadmill exercise; (ii) a study evaluating the pharmacokinetics of recombinant FIX (rFIX) in hemophilia B mice and based on these data; (iii) the treatment study, which tested therapeutic intervention with rFIX. At termination of the treadmill studies the presence of bleeds was evaluated. RESULTS: Treadmill exercise resulted in a high incidence of muscle bleeds in F9-KO mice but not in C57BL/6NTac mice. Treating hemophilia B mice with rFIX before treadmill exercise prevented muscle bleeds. CONCLUSION: A novel model of muscle bleeds in hemophilia B mice, responsive to rFIX, has been developed.

Selection of suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using quantitative real-time polymerase chain reaction.

Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a sensitive technique for quantifying gene expression. Stably expressed reference genes are necessary for normalization of RT-qPCR data. Only a few articles have been published on reference genes in canine tumours. The objective of this study was to demonstrate how to identify suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using RT-qPCR. Primer pairs for 17 potential reference genes were designed and tested in archival tumour biopsies from six dogs. The geNorm algorithm was used to analyse the most suitable reference genes. Eight potential reference genes were excluded from this final analysis because of their dissociation curves. ß-Glucuronidase (GUSB) and proteasome subunit, beta type, 6 (PSMB6) were most stably expressed with an M value of 0.154 and a CV of 0.053 describing their average stability. We suggest that choice of reference genes should be based on specific testing in every new experimental set-up.

Intravenous magnesium does not influence the activity of the coagulation cascade.

Experimental arterial thrombus formation is reduced during intravenous magnesium infusion. It is well documented that magnesium reduces platelet reactivity, but the antithrombotic effect could also originate from anticoagulant properties or increased fibrinolysis. We therefore evaluated the effect of intravenous magnesium on prothrombin fragment 1 + 2 (F1 + 2), thrombin-antithrombin III complex (TAT) concentrations, and fibrin degradation products (FbDP) in a randomized, cross-over study in 14 healthy volunteers. Citrated blood samples were collected at 0, 30, and 180 min. An additional in vitro study on magnesium's effect on the activity of different coagulation factors was carried out. A transient increase was seen in F1 + 2 and TAT after 30 min but without any significant difference between the placebo and magnesium period. FbDP did not change significantly between the two treatments. Increasing concentrations of magnesium dose-dependently decreased binding of activated factor X to activated factor VII (FVIIa), but the decrease was slight and probably without any significance for coagulation at the concentrations tested. No effect was observed on the activity of FVIIa or activated factor VIII. In conclusion, no significant differences were observed on markers of coagulation or fibrinolytic activity during intravenous magnesium infusion. These results indicate that the observed antithrombotic effect of magnesium is more likely to arise from the previously observed platelet inhibition.