Reference interval for the C-ACT activated clotting time test in healthy dogs using a water bath and manual assessment of clot formation.

AIMS: To establish a reference range for the canine C-ACT activated clotting time (ACT) test using a water bath and visual clot assessment technique. METHODS: Healthy, privately owned dogs (n = 48) were prospectively recruited to the study. Blood samples were collected via direct jugular venipuncture for complete blood count, serum biochemistry analysis and measurement of prothrombin time (PT) and activated partial thromboplastin time (aPTT). Five animals with major abnormalities or who became agitated during phlebotomy were excluded. For the 43 remaining animals, 2 mL of blood was collected via the cephalic vein and added directly to a C-ACT tube that was shaken vigorously before being placed in a water bath at 37°C. Tubes were visually assessed for clot formation and C-ACT was recorded in seconds when the magnet within the tube lodged in the clot. RESULTS: The nonparametric reference interval (capturing the central 95% of the data) was 50-80 seconds, with a 90% CI for the lower limit of 50-55 seconds and a 90% CI for the upper limit of 75-80 seconds. The C-ACT ACT test had a positive correlation with aPTT (0.42; 95% CI = 0.13-0.64). There was no evidence of a correlation between C-ACT ACT and age, weight, PT, haematocrit, white blood cell count, platelet count or total protein. CONCLUSIONS AND CLINICAL RELEVANCE: The results of this study suggest that the normal reference interval for ACT in dogs using C-ACT tubes in a 37°C water bath is 50-80 seconds. Care should be taken extrapolating the results of this study to the general population, as the smaller study design had less control for confounders than a larger study. However, when using the described analytical methods, C-ACT tube ACT test results >80 seconds should be considered prolonged in dogs and should prompt further investigation.

Assessment of the CoaguChek-XS portable prothrombin time point-of-care analyzer for horses.

Coagulopathies in horses are common and potentially life-threatening. In equine field medicine, a portable point-of-care (POC) prothrombin time (PT) testing device could be useful to identify early changes in extrinsic clotting. The CoaguChek-XS (Roche Diagnostics) is a small, portable POC PT analyzer used in human medicine. Our preliminary study assessed the suitability of CoaguChek-XS for testing PT in horses and established the PT reference interval (PT RI) in healthy horses using this instrument. Blood samples collected from 102 healthy and ill horses were analyzed with the CoaguChek-XS and compared to a semi-automated coagulometric analyzer (SACA) as the gold standard. There was a significant positive correlation between the 2 measurement methods ( r = 0.765, p < 0.01), and very good agreement, with 97% of the samples falling within limits of agreement. The mean CoaguChek-XS PT coefficient of variation was 0.8%, indicating high precision. With high precision and good agreement with the coagulometric PT, the CoaguChek-XS should be further validated for PT measurement in horses.

In vitro assessment of quality of citrate-phosphate-dextrose-adenine-1 preserved feline blood collected by a commercial closed system.

BACKGROUND: Optimal procedure for storage of feline blood is needed. Open-collection systems have been employed in feline medicine, thus limiting the possibility for storage. OBJECTIVES: To evaluate indicators of quality of feline blood stored for 35 days at +4°C in a closed-collection system specifically designed for cats. ANIMALS: Eight healthy adult European domestic shorthair cats with a weight of 5-6.8 kg. METHODS: This is a case series study. A bacteriological test, CBC, blood smear, pH, osmotic fragility, 2,3-diphosphoglycerate (2,3-DPG), and adenosine triphosphate (ATP) measurement were performed weekly on whole blood (WB) units from day 1 to day 35 after donation. The hemolysis index, lactate and potassium concentrations, prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen were measured on plasma aliquots. RESULTS: One out of eight blood units (BUs) had bacterial growth (Serratia marcescens) at day 35. No significant differences were found regarding CBC, morphology, pH, and osmotic fragility. Despite high inter-individual variability and low starting levels, significant decreases in the mean concentrations of 2,3-DPG (T0 1.99 mmol/g Hb, SD 0.52, T35 1.25 mmol/g Hb, SD 1.43; P = .003) and ATP (T0 1.45 mmol/g Hb, SD 0.71, T35 0.62 mmol/g Hb, SD 0.51; P < .001) were detected during the study, as opposed to an increase in hemolysis (T0 0.11 mmol/L, SD 0.07, T35 0.84 mmol/L, SD 0.19; P < .001), lactate (T0 3.30 mmol/L, SD 0.86, T35 13.36 mmol/L, SD 2.90; P < .001), and potassium (T0 3.10 mmol/L, SD 0.21, T35 4.12 mmol/L, SD 0.35; P < .001) concentrations. CONCLUSIONS AND CLINICAL IMPORTANCE: The commercial BU kit is appropriate for blood collection and conservation of WB in cats. The maintenance of WB quality indicators during storage is essential for future improvements of feline transfusion medicine.

Assessment of hemostatic changes in a model of acute hemorrhage in dogs.

OBJECTIVE: To evaluate hemostatic changes following experimental acute hemorrhage in dogs using traditional coagulation tests (eg, platelet count, prothrombin time [PT], and activated partial thromboplastin time [aPTT]), kaolin-activated thromboelastography (TEG), and whole blood multiple electrode impedance platelet aggregometry. DESIGN: Prospective study. SETTING: Research laboratory. ANIMALS: Five Beagles. INTERVENTIONS: Dogs were anesthetized prior to obtaining blood samples for baseline PCV, total plasma protein (TPP), arterial blood-gas, platelet count, PT, aPTT, TEG, fibrinogen, and aggregometry. Blood was obtained at 4 additional time points, following 20% blood volume loss, 40% blood volume loss, 60 minutes of sustained hypotension, and after autologous blood transfusion. In addition, heart rate and direct arterial blood pressure were measured at each time point. MEASUREMENTS AND MAIN RESULTS: Significant decreases were noted for PCV (P = 0.048), TPP (P < 0.0001), and arterial blood pressures (P < 0.0001) over time. Platelet count did not change significantly (P = 0.879), but platelet function was decreased following hemorrhage when arachidonic acid (P = 0.004) and ADP (P = 0.008) were used as agonists. The TEG variables R (P = 0.030), MA (P = 0.043), and G (P = 0.037) were significantly, albeit mildly, changed following hemorrhage. Significant prolongations in PT (P < 0.0001) and aPTT (P = 0.041), and decreases in fibrinogen concentration (P = 0.002) were also seen. CONCLUSION: Platelet dysfunction occurred following hemorrhage in this model, despite a stable platelet count. Additionally, significant changes associated with hemorrhage were documented in aPTT, fibrinogen, and MA. Platelet function testing in dogs with naturally occurring hemorrhage warrants further investigation.

Assessment of the relationships among coagulopathy, hyperfibrinolysis, plasma lactate, and protein C in dogs with spontaneous hemoperitoneum.

OBJECTIVE: To relate coagulation and fibrinolysis derangements to shock severity as reflected by plasma lactate concentrations in dogs with spontaneous hemoperitoneum (SHP) and determine the impact on transfusions. DESIGN: Prospective, observational, case-control study. SETTING: Three veterinary teaching hospitals. ANIMALS: Twenty-eight client-owned dogs with SHP and 28 breed- and age-matched control dogs. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Blood samples for platelet counts, coagulation, and anticoagulant assays (prothrombin time, activated partial thromboplastin time, fibrinogen, antithrombin, and protein C, thromboelastography [TEG]), fibrinolysis testing (d-dimer and TEG lysis parameters with and without the addition of 50 U/mL of tissue plasminogen activator [TEG LY30 measured with the addition of 50 U/mL of tPA to the blood sample, LY3050 and TEG LY60 measured with the addition of 50 U/mL of tPA to the blood sample, LY6050 ; LY30 and LY60]), and plasma lactate as an indicator of severity of shock were collected from SHP dogs at the time of diagnosis. SHP dogs were hypocoagulable (prolonged prothrombin time and activated partial thromboplastin time, decreased TEG maximum amplitude) and hyperfibrinolytic (increased LY3050 and TEG LY6050 ) compared to controls. The severity of hypocoagulability was related to protein C activity, while the severity of hyperfibrinolysis was related to plasma lactate concentration. Among the 18 dogs discharged from the hospital, LY3050 was significantly associated with the dose of fresh frozen plasma administered, but none of the parameters were associated with the dose of red blood cells administered. CONCLUSIONS: Dogs with SHP have evidence of hypocoagulability, protein C deficiency, and hyperfibrinolysis. Parameters of hyperfibrinolysis were related to plasma lactate concentrations and volume of plasma transfused during hospitalization. These derangements resemble those found in people with acute coagulopathy of trauma and shock, and activation of protein C may be a common feature to both syndromes.

Assessment of a portable prothrombin time analyzer (CoaguChek-XS) in dogs.

OBJECTIVES: To assess the performance of a portable prothrombin time (PT) analyzer (CoaguChek-XS) in dogs. ANIMALS: Ninety-seven dogs composed of 20 healthy dogs, 45 ill dogs, and 32 ill dogs with altered coagulation. PROCEDURES: Samples were concurrently obtained and PT was measured using the CoaguChek-XS and an automated coagulation analyzer. Agreement between methods was assessed using Spearman's correlation and Bland-Altman analysis. RESULTS: The reference interval for PT using the CoaguChek-XS was 9.6-11.5 s. The CoaguChek-XS was easy to use, required a small volume of whole blood, and gave results within 1 min. The mean difference in PT between the 2 methods was 2.58 s (SD 3.10), and 94% of the samples fell within limits of agreement. The correlation was moderate, but significant (r = 0.35, P < 0.001). Sensitivity and specificity of the CoaguCheck-XS PT compared to analyzer PT were 92% and 56%, respectively, and increased to 95% and 77% on intent to treat basis. The coefficient of variance was 0.72%. The CoaguChek-XS identified all 8 dogs with anticoagulant rodenticide intoxication, although a discrepancy was noted from the analyzer results. In anemic dogs (PCV≤25%), the CoaguChek-XS did not display accurate results. CONCLUSIONS: The CoaguChek-XS is a simple, user-friendly, highly precise PT analyzer. Results had moderate correlation and good agreement with a standard method. It can be used reliably for screening dogs when the PT is normal. However, when the CoaguChek-XS PT is prolonged or when the PCV is ≤25%, results should be confirmed using a standard method.

Assessment of prothrombin time, activated partial thromboplastin time, and fibrinogen concentration on equine plasma samples following different storage conditions.

The aim of the present study was to assess the effect of different storage conditions on prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen concentration in clinical samples from healthy horses. A total of 100 healthy horses of varying breeds and gender, ranging in age from 4 to 18 years, with a mean body weight of 480 +/- 70 kg, were used. Blood was collected by jugular venipuncture, and a hemochrome-cytometric examination was conducted on all samples. All blood samples were centrifuged and divided into 4 different aliquots to assess clotting parameters by means of a coagulometer. The first aliquots were analyzed 1 hr after collection, the second aliquots were refrigerated at 8 degrees C for 6 hr, the third aliquots were frozen at -20 degrees C for 24 hr, and the fourth aliquots were frozen at -20 degrees C for 48 hr. Significant differences (P < 0.05) were determined by one-way analysis of variance with repeated measures, and statistical analysis showed a significant effect of the experimental conditions on all parameters studied. In particular, the results demonstrated that coagulation tests can be done within 6 hr when samples are stored at 8 degrees C because the short-term refrigeration does not change the result of analyses; storage at -20 degrees C is acceptable only after 24 hr for PT, aPTT, and fibrinogen measurements because after 48 hr, freezing alters the values of clotting parameters. Therefore, the results of this investigation indicate that clotting parameters remain stable only up to 24 hr in horses without adversely affecting hemostasis test results.

Assessment of changes in hemostatic markers in Cavalier King Charles Spaniels with myxomatous mitral valve disease.

OBJECTIVE: To evaluate markers of hemostasis and their relationship to the degree of mitral regurgitation (MR) and platelet function in Cavalier King Charles Spaniels (CKCSs) with myxomatous mitral valve disease. ANIMALS: 76 clinically healthy CKCSs and 24 control dogs. PROCEDURE: All dogs underwent echocardiographic examination; various hemostatic, hematologic, and biochemical variables were evaluated in blood. The CKCSs were allocated to 1 of 3 groups on the basis of MR severity. In 8 control dogs and 8 CKCSs, plasma von Willebrand factor (vWF) multimer analysis was performed. RESULTS: Compared with control dogs, plasma fibrinogen concentration was higher in all CKCSs and related to left ventricular end diastolic diameter and left atrial-to-aortic root ratio among all CKCSs. The activated partial thromboplastin times and plasma D-dimer concentration were similar among the 4 groups. Plasma vWF concentration was lower in CKCSs with moderate to severe MR, compared with that of CKCSs with no MR and control dogs. There was a relationship between plasma vWF concentration and platelet function in CKCSs but not in control dogs. In 4 CKCSs with moderate to severe MR and low plasma vWF concentration, amounts of vWF high-molecular-weight multimers (HMWMs) were low. CONCLUSIONS AND CLINICAL RELEVANCE: In CKCSs, MR appeared to be associated with a low plasma vWF concentration and likely a loss of vWF HMWMs (possibly through their destruction via shear stress to the blood). The importance of the changes in plasma fibrinogen concentration and the thromboembolic risk in dogs with MR remain to be investigated.

Assessment of factor V, VII and X activities, the key coagulant proteins of the tissue factor pathway in poultry plasma.

1. Assay methods were developed for key components of the tissue factor pathway of blood coagulation, namely Factor V, Factor VII and Factor X. Using these assays, plasma from healthy laying hens, cockerels and broilers was shown to contain functional and equivalent amounts of each of these clotting factors. 2. The plasma activities for Factor V, Factor VII and Factor X can only be accurately determined when chicken tissue factor is used to initiate the coagulation mechanism in poultry plasma. Neither human tissue factor nor rabbit tissue factor forms a fully functional enzyme reactive complex with chicken Factor VII. 3. The overall tissue factor pathway coagulation mechanism was evaluated in plasma from laying hens, cockerels and broilers using the one-stage prothrombin time assay. As long as sufficient tissue factor was used, the overall clotting time results obtained with human recombinant tissue factor were not significantly different from those obtained with chicken tissue factor. 4. We conclude that poultry plasma does possess a fully functional tissue factor coagulation mechanism, but homologous chicken tissue factor must be used for in vitro assays of the components of this pathway.

Assessment of the potential toxicity of a poison for rabbits, pindone (2-pivalyl 1, 3 indandione), to domestic animals.

The toxicity of pindone, a rabbit poison, to horses, cattle, goats, chickens, dogs and cats was investigated, using extension of prothrombin time (PT) as an index of poisoning. The daily dose of pindone, administered for 5 days, ranged from 0.3 mg/kg for dogs to 2.5 mg/kg for chickens. This range of dose rates was considered to be indicative of the worst possible case that could arise following a campaign of baiting for rabbits. Although significant elevations in PT (more than double baseline values) were noted in all species other than horses, clinical signs of anticoagulant poisoning were not observed in any of the species tested. From the observed PT, cattle and cats appeared to be the most susceptible, and horses the least susceptible, to pindone toxicity. The half-lives of the elevated PT were calculated as 3.1 days for cattle, 2.8 days for goats and chickens, 1.9 days for horses and dogs and less than one day for cats. It is proposed that these half-lives can be used as a guide for determining the duration of treatment of pindone-affected animals.