OBJECTIVE To compare stability of hemostatic proteins in canine fresh-frozen plasma (FFP) thawed with a modified commercial microwave warmer (MCM) or warm water bath (37°C; WWB) or at room temperature (22°C).
SAMPLE Fresh-frozen plasma obtained from 8 canine donors of a commercial blood bank.
PROCEDURES A commercial microwave warmer was modified with a thermocouple to measure surface temperature of bags containing plasma. The MCM and a WWB were each used to concurrently thaw a 60-mL bag of plasma obtained from the same donor. Two 3-mL control aliquots of FFP from each donor were thawed to room temperature without use of a heating device. Concentrations of hemostatic proteins, albumin, and D-dimers; prothrombin time (PT); and activated partial thromboplastin time (aPTT) were determined for all samples.
RESULTS Significant decreases in concentrations of factors II, IX, X, XI, fibrinogen, von Willebrand factor, antithrombin, protein C, and albumin and significant increases in PT and aPTT were detected for plasma thawed with the MCM, compared with results for samples thawed with the WWB. Concentrations of factors VII, VIII, and XII were not significantly different between plasma thawed with the MCM and WWB. Concentrations of D-dimers were above the reference range for all thawed samples regardless of thawing method. No significant differences in factor concentrations were detected between control and WWB-thawed samples.
CONCLUSIONS AND CLINICAL RELEVANCE Significant differences in hemostatic protein concentrations and coagulation times were detected for plasma thawed with an MCM but not between control and WWB-thawed samples. Clinical importance of these changes should be investigated.
OBJECTIVE To determine whether critically ill dogs had increased platelet activation and whether the proportion of activated platelets correlated with severity of illness.
ANIMALS 82 dogs in the intensive care unit of a veterinary teaching hospital and 24 healthy control dogs.
PROCEDURES Flow cytometry with monoclonal mouse anti-human CD61 and CD62 antibodies in resting and ADP-treated samples and kaolin-activated thromboelastography were used to compare platelet activation in blood samples of critically ill and control dogs. Serum antithrombin, von Willebrand factor, fibrinogen, and activated protein C concentrations; prothrombin time (PT); and activated partial thromboplastin time (aPTT) were measured. Revised survival prediction index, acute patient physiology and laboratory evaluation, systemic inflammatory response syndrome, and multiple organ dysfunction syndrome scores were used to estimate severity of illness. Severity of illness scores and platelet activation measurements were compared with survival time and duration and cost of hospitalization.
RESULTS Critically ill and control dogs had no differences in platelet activation for non–ADP-treated samples measured. Critically ill dogs had significantly increased platelet activation in response to 2, 6, and 10μM ADP. Critically ill dogs had significantly increased maximum amplitude, α angle, and global clot strength and significantly decreased clot formation time. Critically ill dogs had significantly increased fibrinogen concentration, PT, and aPTT and significantly decreased antithrombin concentration. Survivors and nonsurvivors had similar flow cytometry and thromboelastography values. Three dogs developed macrothrombosis.
CONCLUSIONS AND CLINICAL RELEVANCE In this study, critically ill dogs had hyperreactive platelets, which may have contributed to a high incidence of hypercoagulability in this patient population.
OBJECTIVE To evaluate the degree of activation of the contact pathway in citrated equine whole blood over holding times ≤ 30 minutes and assess effects of contact activation on recalcification-initiated thromboelastometry.
ANIMALS 11 healthy adult mixed-breed horses.
PROCEDURES Blood was collected by atraumatic jugular venipuncture into prewarmed evacuated siliconized glass tubes containing citrate anticoagulant and held at 37°C for ≤ 30 minutes. Thromboelastometry was performed with an in vitro viscoelasticity (thromboelastometry) monitoring system. Factor XII and factor XI procoagulant activities were determined in contemporaneously collected platelet-poor plasma samples by assessing changes in turbidity for 1 hour at approximately 25°C, with clotting times calculated by fitting a line to the steepest segment of the absorbance curve and determining its intersection with baseline. Effect of holding time on thromboelastometry parameters and plasma enzyme activity was evaluated by repeated-measures ANOVA on ranks. Association of procoagulant activities with coagulation time was determined by Spearman rank-order correlation analysis.
RESULTS Thromboelastometry parameters (coagulation time, clot formation time, α angle, and maximum clot firmness) reflected significant increases in coagulability during the holding period. Factor XII and factor XI procoagulant activities were significantly increased at 30 minutes, compared with 2 or 10 minutes (indicating contact activation of samples), and had significant negative correlation with coagulation time.
CONCLUSIONS AND CLINICAL RELEVANCE Ex vivo activation of the contact system in equine whole blood was evident, suggesting that recalcification of blood in the absence of a trigger is not an acceptable method of assessing the hemostatic system in horses.
OBJECTIVE To evaluate canine erythrocyte concentrates (ECs) for the presence of procoagulant phospholipid (PPL), determine whether PPL concentration changes during the course of storage of ECs, and ascertain whether prestorage leukoreduction (removal of leukocytes via gravity filtration) reduces the development of PPL.
SAMPLE 10 whole blood units (420 g each) collected from 10 random-source, clinically normal dogs (1 U/dog).
PROCEDURES The dogs were randomized to 1 of 2 groups. Of the 10 whole blood units collected, 5 were processed through a standard method, and 5 underwent leukoreduction. Whole blood units were processed to generate ECs, from which aliquots were aseptically collected from each unit weekly for 5 weeks. Supernatants from the concentrates were evaluated for procoagulant activity, which was converted to PPL concentration, by use of an automated assay and by measurement of real-time thrombin generation.
RESULTS Supernatants from stored canine ECs contained procoagulant activity as measured by both assays. In general, the PPL concentration gradually increased during the storage period, but leukoreduction reduced the development of increased procoagulant activity over time.
CONCLUSIONS AND CLINICAL RELEVANCE The presence of PPL in canine ECs may be associated with procoagulant and proinflammatory effects in vivo, which could have adverse consequences for dogs treated with ECs.
Objective—To develop a flow cytometric assay to quantify platelet-derived microparticles (PMPs) in equine whole blood and plasma.
Sample—Citrate-anticoagulated whole blood from 30 healthy adult horses.
Procedures—Platelet-poor plasma (PPP) was prepared from fresh whole blood by sequential low-speed centrifugation (twice at 2,500 × g). Samples of fresh whole blood and PPP were removed and stored at 4° and 24°C for 24 hours. Platelet-derived microparticles were characterized in fresh and stored samples on the basis of the forward scatter threshold (log forward scatter < 101) and labeling with annexin V (indicating externalized phosphatidylserine) and CD61 (a constitutive platelet receptor). A fluorescent bead–calibrated flow cytometric assay was used to determine microparticle counts. Platelet counts, prothrombin time, and activated partial thromboplastin time were measured in fresh samples.
Results—Significantly more PMPs were detected in fresh whole blood (median, 3,062 PMPs/μL; range, 954 to 13,531 PMPs/μL) than in fresh PPP (median, 247 PMPs/μL; range, 104 to 918 PMPs/μL). Storage at either temperature had no significant effect on PMP counts for whole blood or PPP. No significant correlation was observed between PMP counts and platelet counts in fresh whole blood or PPP or between PMP counts and clotting times in fresh PPP.
Conclusions and Clinical Relevance—Results indicated that the described PMP protocol can be readily used to quantify PMPs in equine blood and plasma via flow cytometry. Quantification can be performed in fresh PPP or whole blood or samples stored refrigerated or at room temperature for 24 hours.
Objective—To optimize the overall hemostasis potential (OHP) assay for use with canine platelet-poor plasma and determine reference intervals in healthy dogs.
Animals—40 healthy dogs.
Procedures—Blood was collected from the dogs into citrated tubes, and platlet-poor plasma was obtained. The OHP assay and standard coagulation assays (prothrombin time, activated partial thromboplastin time, and fibrinogen concentration) were performed for each sample. The OHP assay outputs were tested for correlations with results of the standard coagulation assays, age, and sex.
Results—Modifications to the published methodology for the OHP assay were required for use with canine plasma, with less coagulation activator (thrombin) and more fibrinolysis activator (tissue plasminogen activator) than used with human plasma. Male dogs had a higher OHP than did females. High fibrinogen concentrations were associated with increases in maximum optical density, OHP, and overall coagulation potential, and reduced prothrombin time was associated with increases in maximum optical density, overall coagulation potential, OHP, and maximum slope.
Conclusions and Clinical Relevance—Results supported the use of the OHP assay as an accessible, cost-effective global coagulation assay. Further research is required to determine its clinical application as an alternative to thromboelastography or thrombin generation assays.
Objective—To determine the effect of large colon ischemia and reperfusion on concentrations of the inflammatory neutrophilic protein calprotectin and other clinicopathologic variables in jugular and colonic venous blood in horses.
Animals—6 healthy horses.
Procedures—Horses were anesthetized, and ischemia was induced for 1 hour followed by 4 hours of reperfusion in a segment of the pelvic flexure of the large colon. Blood samples were obtained before anesthesia, before induction of ischemia, 1 hour after the start of ischemia, and 1, 2, and 4 hours after the start of reperfusion from jugular veins and veins of the segment of the large colon that underwent ischemia and reperfusion. A sandwich ELISA was developed for detection of equine calprotectin. Serum calprotectin concentrations and values of blood gas, hematologic, and biochemical analysis variables were determined.
Results—Large colon ischemia caused metabolic acidosis, a significant increase in lactate and potassium concentrations and creatine kinase activities, and a nonsignificant decrease in glucose concentrations in colonic venous blood samples. Values of these variables after reperfusion were similar to values before ischemia. Ischemia and reperfusion induced activation of an inflammatory response characterized by an increase in neutrophil cell turnover rate in jugular and colonic venous blood samples and calprotectin concentrations in colonic venous blood samples.
Conclusions and Clinical Relevance—Results of this study suggested that large colon ischemia and reperfusion caused local and systemic inflammation in horses. Serum calprotectin concentration may be useful as a marker of this inflammatory response.
Objective—To determine the degree of agreement between 2 analyzers for measurement of total CO2 concentration (ctCO2) in equine plasma.
Animals—6 healthy untrained horses, 6 trained Standardbreds undergoing a simulated race protocol, and 135 trained Standardbreds at a racetrack.
Procedures—Jugular venous blood samples were obtained from all horses. Two analyzers (commonly used analyzer A and less expensive analyzer B) were used to measure plasma ctCO2 in each sample. Validation of both analyzers was conducted in accordance with guidelines established by the Clinical and Laboratory Standards Institute and involved characterization of linearity, total analytic error, and bias estimation.
Results—Total analytic error (instrument SD) was 0.58 mmol/L (coefficient of variation, 1.6%) and 0.49 mmol/L (coefficient of variation, 1.4%) for analyzers A and B, respectively, when measuring an aqueous standard containing 36.0 mmol of CO2/L. A 1 g/L decrease in plasma protein concentration corresponded to an increase in ctCO2 measured with analyzer B of 0.065 mmol/L. A difference plot indicated that analyzer B produced values 2.7% higher than analyzer A for 103 samples from the 6 trained and exercised Standardbreds (mean plasma protein concentration, 67 g/L).
Conclusions and Clinical Relevance—Analyzer B provided adequate precision and linearity for measurement of ctCO2 from 5 to 40 mmol/L and was therefore suitable for measuring ctCO2 in equine plasma, provided allowances are made for changes in plasma protein concentration.
Objective—To assess a commercially available point-of-care assay for measurement of bovine cardiac troponin I (cTnI) concentration in blood and plasma samples.
Sample—Prepared bovine plasma standard samples with known concentrations (0 to 1.0 ng/mL) of cTnI and blood and plasma samples obtained from 28 healthy 2.5-month-old Holstein calves.
Procedures—Coefficients of variation were calculated for concentrations of cTnI in prepared standards determined with the point-of-care assay, and values were compared with the known concentrations. The cTnI concentrations in blood samples obtained from calves determined with the point-of-care assay were compared with cTnI concentrations in plasma samples obtained from those animals determined with a validated immunoassay.
Results—The coefficients of variation of cTnI concentrations determined for prepared standards by use of the point-of-care assay were low (< 20%) for standards with cTnI concentrations ≥ 0.025 ng/mL. The blood cTnI concentrations determined with the point-of-care assay were not significantly different from the plasma cTnI concentrations determined with the validated immunoassay.
Conclusions and Clinical Relevance—Results of this study indicated the point-of-care assay had high precision for determination of cTnI concentrations in most evaluated prepared bovine plasma standard samples. The point-of-care assay may be useful for determination of circulating concentrations of cTnI in cattle.
Objective—To evaluate fecal calprotectin concentrations in healthy dogs and dogs with chronic diarrhea, to identify cutoff values for fecal calprotectin concentrations for use in differentiating dogs with chronic diarrhea and a canine chronic enteropathy clinical activity index (CCECAI) < 12 from dogs with chronic diarrhea and a CCECAI ≥ 12, and to evaluate the association between histologic evidence of intestinal mucosal changes and fecal calprotectin concentrations in dogs with chronic diarrhea.
Sample—Fecal samples from 96 adult dogs (27 dogs with chronic diarrhea and 69 healthy control dogs).
Procedures—Severity of clinical signs was evaluated on the basis of the CCECAI scoring system. Endoscopy was performed in all dogs with chronic diarrhea, and mucosal biopsy specimens were evaluated histologically. Fecal calprotectin concentration was quantified via radioimmunoassay.
Results—Fecal calprotectin concentrations were significantly higher in dogs with chronic diarrhea than in healthy control dogs. Fecal calprotectin concentrations were also significantly higher in dogs with a CCECAI ≥ 12, compared with concentrations for dogs with a CCECAI between 4 and 11. Fecal calprotectin concentrations were significantly higher in dogs with chronic diarrhea associated with histologic lesions, compared with concentrations in control dogs, and were significantly correlated with the severity of histologic intestinal lesions. Among dogs with chronic diarrhea, the best cutoff fecal calprotectin concentration for predicting a CCECAI ≥ 12 was 48.9 μg/g (sensitivity, 53.3%; specificity, 91.7%).
Conclusions and Clinical Relevance—Fecal calprotectin may be a useful biomarker in dogs with chronic diarrhea, especially dogs with histologic lesions.