Validation of human recombinant tissue factor-activated thromboelastography on citrated whole blood from clinically healthy dogs

BACKGROUND: Thromboelastography (TEG) is an analytical method that enables global assessment of hemostatic function in whole blood (WB) with evaluation of both plasma and cellular components of hemostasis. TEG has a largely unused potential in the diagnostic workup and monitoring of dogs with hemostatic disorders and it may be a valuable supplement to traditional coagulation parameters. OBJECTIVES: The objective of this study was to establish a clinically applicable reference interval for a TEG assay using recombinant human tissue factor (TF) as the activator on citrated WB from clinically healthy dogs and to evaluate the stability of citrated WB stored for 30 minutes (T30) and 120 minutes (T120) at room temperature (RT). Additionally, we evaluated the analytical variation in reaction time (R), clotting time (K), angle (alpha), and maximum amplitude (MA). METHODS: Blood was collected from 18 clinically healthy dogs. Duplicate TEG analyses with TF as the activator at a concentration of 1:50,000 were performed on canine citrated WB at T30 and T120. R, K, a, and MAwere analyzed. RESULTS: Mean TEG values at T30/T120 were R = 5.61/4.91 minutes, K = 4.20/3.34 minutes, alpha = 45.33/50.90 degrees , and MA = 47.96/50.19 mm. Significant differences in these values were observed after storage for T30 and T120 at RT, with a tendency towards hypercoagulability at T120. The mean coefficients of variation were low. CONCLUSIONS: Canine citrated WB can be used for TEG analysis with human recombinant TF as the activator when stored at RT for T30 or T120. At both time points, the analytical variation was low, suggesting that TEG analysis may be of value in evaluating dogs with hemostatic disorders. A fixed time point should be chosen for serial measurements.     

Comparison of Axillary And Heating Block Methods of Activated Clotting Time (ACT) in Dogs

The activated clotting time (ACT) is commonly used in veterinary medicine as an assessment of dysfunction within the intrinsic clotting cascade. Performing the test requires little techincal expertise and no special equipment except for a heating block or constant temperature waterbath, neither of which is routinely found in veterinary practices. The purpose of this study was to determine whether performing the test using a human axilla as the heat source was accurate for both normal dogs and clinically ill dogs (with prolonged ACT's) when compared to using a heating block as the head source. The results of this study reveal that the axillary method of ACT determination has acceptable clinical agreement with the heating block method. Thus, the axillary method of ACT determination is an acceptable alternative when no constant temperature heating source is available.     

Use of whole blood for spectrophotometric determination of cholinesterase activity in dogs

Whole blood has been compared with erythrocytes and plasma for spectrophotometric cholinesterase determination in the dog. Cholinesterase activity was characterized using two substrates: acetylthiocholine and butyrylthiocholine. Acetylcholinesterase was the only form of cholinesterase present on erythrocytes and hydrolysed only acetylthiocholine. Butyrylcholinesterase (pseudocholinesterase) was predominant in plasma, hydrolysing mainly butyrylthiocholine. Based on these results, a method based on the use of two substrates (acetylthiocholine for monitoring acetylcholinesterase and butyrylthiocholine for determining butyrylcholinesterase) in the same whole blood sample is recommended for canine cholinesterase analysis. This way of monitoring both enzymes can be easily automated, yielding good within (CVs < 5%) and between-run (CVs < 7%) precision.     

In vitro heparinization of canine whole blood with low molecular weight heparin (dalteparin) significantly and dose‐dependently prolongs heparinase‐modified tissue factor‐activated thromboelastography parameters and prothrombinase‐induced clotting time

Background: Low‐molecular‐weight heparin (LMWH) is being used increasingly in veterinary medicine for both treatment and prophylaxis of thromboembolic disease, but no predictable patient‐side method exists to monitor its effect. Objectives: The aim of this study was to evaluate thromboelastography (TEG) and prothombinase‐induced clotting time (PiCT) assays for detecting hemostatic alterations following in vitro heparinization of canine whole blood with dalteparin (Fragmin). Methods: Citrated whole‐blood samples were collected from 7 clinically healthy dogs. Dalteparin was added at concentrations of 0, 0.156, 0.625, 1.25, and 2.5 U/mL of whole blood. TEG was performed using heparinase cups with tissue factor (TF, 1:50,000) and kaolin as activators. Reaction time (R), clotting time (K), angle (α), and maximum amplitude (MA) were recorded. PiCT and anti‐FXa activity were measured in plasma. Results: With TF, increasing concentrations of dalteparin significantly prolonged R and K and significantly decreased α and MA. K, α, and MA ratios were significantly different from baseline at all dalteparin concentrations and R was significantly different from baseline at concentrations of 0.625, 1.25, and 2.5 U/mL. With kaolin, only R was significantly different from baseline at dalteparin concentrations of 0.625 and 2.5 U/mL. PiCT detected dalteparin concentrations ≤ 0.625 U/mL, with a good linear correlation (r²=.96, P<.0001). Conclusion: These results suggest that TF‐activated TEG and PiCT assays should be further evaluated as promising new methods for evaluating the effect of LMWH, using doses in the recommended clinical range and prospective clinical studies.     

Comparison of radioimmunoassay and chemiluminescent assay methods to estimate canine blood cortisol concentrations

Background Non‐radioactive assay methods are widely used in commercial laboratories to measure canine blood cortisol concentrations, despite a paucity of published validity data of these tests compared with the traditional ‘gold standard’ radioimmunoassay. Objectives To compare a commercial chemiluminescence assay with radioimmunoassay for blood cortisol measurement, determine the effect of storage on the radioimmunoassay, and determine the impact of any differences on clinical decisions. Methods The study included 54 client owned dogs undergoing adrenal function testing. Fresh plasma or serum samples (n = 170) were assayed for cortisol using radioimmunoassay (RIA1). Samples (n = 196) were also frozen and stored in batches, and assayed by chemiluminescence and radioimmunoassay (RIA2). Results Overall, there was a strong correlation (r² = 0.967, P < 0.001) between RIA2 and chemiluminescence concentrations without significant difference between means. Strong correlations were present for RIA2 and chemiluminescence at concentration subgroups of > 400 nmol/L (r² = 0.869, P < 0.001), < 100 nmol/L (r² = 0.790, P < 0.001), and < 40 nmol/L (r² = 0.738, P < 0.001). Significant differences between means were present for RIA2 and chemiluminescence concentrations in the < 100 nmol/L, and < 40 nmol/L (P < 0.001) groups. Despite a significant difference in RIA1 and RIA2 results overall, there was no significant difference between RIA1 and RIA2 for any of the concentration groups. In seven cases, discrepant RIA2 and chemiluminescence results may have altered clinical decisions. Conclusions Although RIA and chemiluminescence cortisol concentrations appear highly correlated, a significant difference may exist for concentrations less than 100 nmol/L in stored canine sera. Results of chemiluminescence cortisol assays should be interpreted with caution unless the specific assay method in the laboratory has been adequately validated in dogs.     

Comparison of whole blood and plasma potassium concentrations in dogs using the Reflovet system

Background: The Reflovet system is designed for chemical analysis of whole blood. However, plasma or serum is recommended for potassium analysis because of possible interference from RBC potassium. Because RBC potassium concentration is low in most canine erythrocytes, however, there should be little or no interference.
Objective: The objective of this study was to compare potassium results obtained in whole blood and in plasma from dogs using the Reflovet system.
Methods: Blood samples were collected from 104 dogs into lithium-heparin tubes. The potassium concentration was measured in whole blood, and subsequently the PCV was measured. Samples were centrifuged and the potassium concentration was measured in plasma. Comparisons were made using Deming's regression and Bland-Altman difference plots.
Results: There was very good correlation between results of potassium measurements in whole blood and plasma ( r = 0.93). Potassium values were moderately lower in whole blood: Potassium_blood= 0.912 × Potassium _plasma+ 0.119. Hemolysis had a negligible effect on the results, but the difference increased with the PCV value. In more than 90% of samples, the difference between the 2 measurements was ≤ 0.3 mmol/L.
Conclusion: There is only a negligible difference in most cases between potassium values in canine plasma and whole blood using the Reflovet system.     

A clinical comparison between a non‐invasive blood pressure monitor using high definition oscillometry (Memodiagnostic MD 15/90 Pro) and invasive arterial blood pressure measurement in anaesthetized dogs

ObjectiveTo compare high definition oscillometry (HDO) to invasive blood pressure measurement in anaesthetized dogs.Study designProspective, clinical trial.AnimalsFifty dogs weighing 1.95–79 kg (mean 23.5 kg).Materials and methodsAnaesthetic and peri–anaesthetic management was chosen according to each dog's physical status and anaesthetist's preference. Direct arterial blood pressure measurements were performed using a catheter placed in the dorsal pedal artery and an electronic pressure transducer connected to a multiparameter monitor. Non–invasive blood pressure measurements were performed using an appropriately sized cuff placed around the tail base. Comparisons between the two methods were made using Bland and Altman plots. The data are reported as mean bias (lower, upper limits of agreement). Further analysis was performed after separating the data into the following categories based on invasive mean arterial blood pressure (MAP): high (MAP > 100 mmHg), medium (70 mmHg < MAP < 100 mmHg) and low (MAP < 70 mmHg) blood pressure (BP). The two methods were compared as used clinically.ResultsEight hundred measurement pairs for invasive and HDO BP readings were compared. Overall, the HDO measured lower values for SAP and DAP but higher for MAP than the invasive method. The lowest bias (upper, lower limits of agreement) were obtained for MAP, ?1 (?22, 19) mmHg. The biggest discrepancy between the methods was reflected by a large bias (limits of agreement) 5 (?34, 45) mmHg, was for SAP. The results for DAP were between those for SAP and MAP with a bias (limits of agreement) of 3 (?20, 27) mmHg. When the values were separated into the pressure range categories the HDO measured higher in the high, medium and low BP groups, with the exception of SAP in the low BP group.ConclusionsWhen considering the mean bias, the accuracy of HDO compared well with direct arterial blood pressure, but the precision was poor, as determined by wide limits of agreement.Clinical relevanceUsing trends and serial measurements rather than a single measurement for clinical decision making is recommended with both methods, when used as reported here.