Preliminary evaluation of reference intervals for a point-of-care viscoelastic coagulation monitor (VCM Vet) in healthy adult horses.

OBJECTIVE: To evaluate a point-of-care viscoelastic coagulation monitor (VCM Vet) for use in horses by assessing variability between devices and establish reference intervals (RIs) for healthy adult horses. DESIGN: Prospective observational study. SETTING: Two university teaching hospitals. ANIMALS: Healthy adult horses (n = 68). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Blood collected by direct jugular venipuncture was applied directly from the syringe into 2 VCM Vet cassettes to establish coefficients of variation (CVs) and RIs for reported parameters of clotting time (CT), clot formation time (CFT), alpha angle, amplitude at 10 and 20 minutes, maximum clot firmness, and lysis index at 30 and 45 minutes. CVs for each parameter were within clinical tolerance. There was a significant difference in CT between institutions (P < 0.001). Differences in CV were found between institutions for CT (P = 0.003) and CFT (P = 0.01). Healthy horse RIs were calculated for the overall data set and each individual institution. Calculated RIs were as follows: CT, 255.6-1233.9 seconds; CFT, 89.4-581 seconds; alpha angle, 11.4-53.6°; maximum clot firmness, 18-37.7; lysis index at 30 minutes, 97.3%-102.1%; lysis index at 45 minutes, 80.8%-103.3%; amplitude at 10 minutes, 8.7-28.3; and amplitude at 20 minutes, 17.4-35.7. CONCLUSIONS: VCM Vet is a repeatable and practical option for rapid point-of-care assessment of hemostasis in horses but has a wide RI and is susceptible to variability. Establishment of institution-specific RIs is recommended.

Comparison of healthy blood donor Greyhounds and non-Greyhounds using a novel point-of-care viscoelastic coagulometer.

OBJECTIVE: To measure and compare viscoelastic coagulation in 2 canine blood donor populations using a novel, point-of-care device (VCM Vet Analyzer, VCM). DESIGN: Cohort study. SETTING: Academic and commercial veterinary blood banks. ANIMALS: Non-Greyhounds from community-based blood donor program and Greyhounds from a blood bank colony. INTERVENTION: Blood was collected from all dogs via direct venipuncture for a complete hemogram, biochemistry, and point-of-care viscoelastic coagulation. MEASUREMENT AND MAIN RESULTS: All biochemical measurements for all dogs in Group NG (n = 38, non-Greyhounds) and Group G (n = 53, Greyhounds) were within local reference intervals. Hematology data showed significant statistical differences between groups in hemoglobin, RBC, platelet, and WBC concentrations. Group G demonstrated lower maximum clot firmness (MCF) with 17 VCM units (26 VCM units in Group NG), increased lysis with 30 VCM units at 30 minutes (LI30) and 27 VCM units at 45 minutes (LI45) (86 VCM units LI30 and 85 VCM units LI45 in Group NG), and decreased amplitude of 13 VCM units 10 minutes (A10) after clot time (CT) and 6 VCM units 20 minutes after CT (A20) (18 VCM units [A10] and 22 VCM units [A20] in Group NG). CONCLUSION: This study found differences between healthy Greyhound and non-Greyhound blood donors in measures of clot strength and fibrinolysis as measured by the VCM. Whereas Greyhound have unique hematologic and hemostatic profiles, these measured viscoelastic differences are important to note prior to and following surgical intervention to aid in clinical decision-making if bleeding complications develop.

An in vitro study of canine cryopoor plasma to correct vitamin K-dependent coagulopathy in dogs.

OBJECTIVE: To compare the efficacy of fresh frozen plasma (FFP) with cryopoor plasma (CPP) to treat vitamin K-dependent factor deficiency in a canine in vitro setting. DESIGN: In vitro laboratory study. SETTING: University veterinary medical teaching hospital. ANIMALS: Seven units of FFP and 6 units of CPP from unique canine donors from the university veterinary blood bank. INTERVENTIONS: Canine FFP was adsorbed by oral barium sulfate suspension to mimic vitamin K-dependent coagulopathy. A sequential mixing study was completed by adding FPP or CPP to the adsorbed plasma. Measurements of prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, and factor activities of factors II, VII, and IX (FII, FVII, and FIX) were compared between the 2 treatment groups. MEASUREMENTS AND MAIN RESULTS: When comparing the sequential addition of CPP or FPP to adsorbed plasma, the following had no statistical significance: PT (P = 0.94), aPTT (P = 0.66), FII (P = 0.05), and FIX (P = 0.90). There was a dose-dependent decrease with PT and aPTT and a dose-dependent increase with FII and FIX. In contrast, after the addition of either CPP or FFP, there was a significant difference between the treatment groups for the concentration of fibrinogen (P = 0.005) and activity of FVII (P = 0.044), with FFP resulting in a greater concentration of fibrinogen and CPP resulting in a greater concentration of FVII. Measurements of factor X (FX) were initially included in the study but were later excluded because FX appeared to be continually adsorbed even after the addition of CPP or FFP. CONCLUSIONS: CPP partially corrected the coagulation times and concentration of vitamin K-dependent coagulation factors to the same degree as FFP. CPP, generally less expensive than FFP, may provide an alternative treatment option for vitamin K-dependent coagulopathies, although in vivo testing is needed.

Establishment of a reference interval for a novel viscoelastic coagulometer and comparison with thromboelastography in healthy cats.

BACKGROUND: Viscoelastic analysis provides information on the dynamics and strength of clot formation as well as clot stability. A novel point-of-care viscoelastic test (Viscoelastic Coagulation Monitor Vet, VCM Vet) could be more cost-effective, simpler to use, and more portable than thromboelastography (TEG). OBJECTIVES: The primary aim of this study was to establish a feline reference interval (RI) for the VCM Vet. A secondary aim was to compare VCM Vet analysis with TEG in healthy cats. METHODS: Fifty-six healthy cats were enrolled in this study. Linear regression was completed to determine whether age and CBC parameters were associated with the VCM Vet parameters and if TEG parameters were correlated with VCM Vet data. Statistical significance was set at P < .05. RESULTS: Fifty-three VCM Vet tracings were used to determine RIs for healthy cats. The determined RIs were: clot time (CT) 104-438 seconds; clot formation time (CFT) 104.5-488 seconds; alpha angle (AA) 30.5°-70°; a10 13.8-32.7 VCM units; a20 19.2-40.1 VCM units; maximum clot formation (MCF) 22.5-44.8 VCM units; Lysis Index 30 (Li30) 92.9%-100.9%; and Lysis Index 45 (Li45) 92%-100%. Linear regression identified a strong positive correlation between the CT and R-time measured using the VCM Vet and TEG methods, respectively; no other parameters were correlated. CONCLUSIONS: The use of VCM Vet is feasible in cats, and we determined the first described feline RIs for this test. In general, the VCM Vet data did not correlate with TEG in healthy cats.

Non-lethal diagnostic methods for koi herpesvirus in koi Cyprinus carpio.

Cyprinid herpesvirus 3, also known as koi herpesvirus (KHV), is a viral pathogen responsible for mass mortalities of carp worldwide. In this study, we compared the sensitivity and specificity of ELISA and quantitative PCR (qPCR) methods for the diagnosis of KHV in experimentally infected koi Cyprinus carpio over an 11 mo period. Koi were exposed to KHV at 18 ± 1°C (permissive temperatures for KHV disease) in laboratory-controlled conditions. At 21 d post challenge, the temperature in the system was decreased to <15°C (non-permissive temperature for KHV disease), and fish were monitored for the following 11 mo. At different time points throughout the study, samples of blood and gills were collected from exposed and control koi and subjected to qPCR and ELISA. Survival proportions of 53.3 and 98.8% in exposed and control treatments, respectively, were recorded at the end of the challenge. Traditional receiver-operating characteristic analysis was used to compare the sensitivity of the ELISA and blood and gill qPCR during permissive and non-permissive temperatures. ELISA was superior to qPCR of gills and whole-blood samples in detecting previous exposure to KHV. Similar results were obtained in a second experiment exposing koi to KHV and inducing persistent infection at >30°C (non-permissive temperature for KHV disease). Finally, KHV ELISA specificity was confirmed using cyprinid herpesvirus 1-exposed koi through a period of 3 mo. This study demonstrates that the combination of ELISA and gill qPCR should be recommended in the diagnosis of KHV exposure of suspected carrier-state fish.

Equine platelet concentrate preparation and validation.

BACKGROUND: Development of equine platelet concentrate (PC) would aid management of cases requiring transfused platelets (PLTs), where adminstration of whole-blood or platelet-rich plasma (PRP) might be contraindicated. OBJECTIVES: To test and validate a method for production of an equine PRP-PC product. ANIMALS: Six healthy Thoroughbred geldings from a research herd. METHODS: In this prospective experimental study, whole blood was collected and processed through multiple centrifugation steps to yield 120 mL of PC. The PC was stored at 22°C and gently and continuously agitated. Measurements of PLT count, pH, and concentrations of glucose, lactate, electrolytes, lactate dehydrogenase (LDH), and aspartate aminotransferase (AST), as well as partial pressures of oxygen and carbon dioxide were performed on days 0, 1, 2, 3, 5, and 7. Platelet aggregometry and bacterial culture were also performed. RESULTS: The PC always had a PLT count of ≥550 × 103 cells/µL. Aggregometry graph amplitude (P < .0001) and area under the curve (P < .05) significantly decreased over time. Sodium, chloride, lactate (P < .0001), and oxygen (P < .01) concentrations significantly increased over time. pH (P < .001), glucose and bicarbonate concentrations (P < .0001) significantly decreased over time. There was no significant difference in potassium concentration, PLT count, LDH and AST activities and no bacterial growth from culture. CONCLUSIONS AND CLINICAL IMPORTANCE: The described technique yielded a PC that meets the standards of the American Association of Blood Banks for human PC.

In vitro assessment of the effect of acidemia on coagulation in dogs.

OBJECTIVE: Minimal data are available assessing the effect of acidemia on coagulation in dogs. The objective of this study was to assess the effect of in vitro acidification of canine blood on coagulation as measured via thromboelastography (TEG) and traditional tests of coagulation. We hypothesized that worsening acidemia would lead to progressive impairment on coagulation. DESIGN: Prospective study. SETTING: University teaching hospital. ANIMALS: Six client-owned dogs. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Blood was collected into 3.2% sodium citrate vacutainer tubes. The pH of blood was adjusted from baseline using hydrochloric acid to create weak acidemia and strong acidemia. Coagulation was assessed using TEG, prothrombin time, and activated partial thromboplastin time. Kruskal-Wallis tests with Dunn's post hoc comparison tests were used to compare groups. Strong acidemia samples were significantly more acidic than baseline based on pH (P < 0.0005), HCO3- (P < 0.0062), pCO2 (P < 0.0001), and base excess (P < 0.0001). Using TEG, in vitro acidification of blood caused significant, progressive impairment of maximum amplitude (P = 0.0282) and alpha angle (P = 0.0312). Acidification of blood had no significant effect on prothrombin time (P = 0.345) or activated partial thromboplastin time (P = 0.944). CONCLUSIONS: In vitro acidification of canine whole blood results in hypocoagulability as measured by some TEG variables.

Comparison of equine platelet function and survival in whole blood collected in acid-citrate-dextrose solution or citrate-phosphate-dextrose-adenine solution.

BACKGROUND: Equine whole blood collection and storage methods have been evaluated to assess red blood cell viability; however, platelet (PLT) viability has not been comprehensively assessed. OBJECTIVES: The purpose of the study was to compare viability of PLTs collected in whole blood into 2 different anticoagulants. METHODS: Whole blood from 6 healthy adult Thoroughbred horses was collected into citrate-phosphate-dextrose-adenine (CPDA) or acid-citrate-dextrose (ACD). Platelet count, pH, and concentrations of glucose, lactate, carbon dioxide, oxygen, bicarbonate, sodium, potassium, and chloride were measured within 10 minutes of collection and then again one hour later at which time PLT aggregometry was performed to assess PLT function. RESULTS: Aggregometry mean amplitudes were significantly higher in CPDA compared to ACD. Blood glucose, pH, bicarbonate, sodium, and lactate concentrations were significantly higher in CPDA compared to ACD. Lactate concentration was higher following one hour in either anticoagulant. Potassium, oxygen, and carbon dioxide concentrations were significantly higher in ACD compared to CPDA at collection. CONCLUSIONS: Platelet aggregometry results suggest that CPDA is superior to ACD for maintaining PLT viability following whole blood collection. This may be associated with the higher, more neutral pH as well as an increase in glucose available for metabolism. Although lactate was increased in the CPDA samples it was not high enough to decrease pH and therefore may not have been high enough to cause morphologic lesions and loss of PLT viability.

Equine bone marrow volume reduction, red blood cell depletion, and mononuclear cell recovery using the PrepaCyte-CB processing system.

BACKGROUND: Volume reduction and RBC depletion of equine bone marrow specimens are necessary processing steps for the immediate therapeutic use of bone marrow (BM)-derived mesenchymal stem cells (MSC), and for MSC expansion in culture. OBJECTIVES: The purpose of the study was to evaluate the ability of the PrepaCyte-CB processing system to reduce volume, deplete RBC, and recover mononuclear cells (MNC) from equine BM specimens. METHODS: One hundred and twenty mL of heparinized BM were obtained from each of 90 horses. A CBC was performed on the BM pre- and post-PrepaCyte-CB processing. Volume and RBC reduction, and total nucleated cell (TNC) and MNC recoveries were determined. RESULTS: Bone marrow volume was reduced from 120 mL to 21 mL with a median RBC depletion of 90.1% (range, 62.0-96.7%). The median preprocessing total TNC count was 2.2 × 10(9) (range, 0.46-7.9 × 10(9)) and the median postprocessing TNC count was 1.7 × 10(9) (range, 0.3-4.4 × 10(9); P < .0001), with a median recovery of 73.5% (range, 22.4-216.7%). The median preprocessing total MNC count was 0.9 × 10(9) (range, 0.1-4.7 × 10(9)) and median postprocessing total MNC count was 0.8 × 10(9) (range, 0.1-2.7 × 10(9); P = .06), with a median recovery of 83.7% (range, 15.4-413.9%). CONCLUSIONS: The PrepaCyte-CB processing system can be used to deplete both volume and RBC, and recover MNC from equine BM specimens. Further studies assessing the viability of MSC and the efficacy of MSC expansion after using the PrepaCyte-CB processing system are warranted.

Comparison of the platelet-rich plasma and buffy coat protocols for preparation of canine platelet concentrates.

BACKGROUND: Platelet (PLT) concentrates (PC) can be produced via the buffy coat (BC) or platelet-rich plasma (PRP) protocols. The 2 methods have not been compared with canine blood. OBJECTIVES: The aims of the study were to compare the PLT, WBC, and RBC concentrations, in vitro PLT function, and markers of platelet storage lesion (PSL) in canine PC generated by 2 different protocols, and determine microbial growth throughout storage. METHODS: PC from 8 healthy donor dogs were produced using 2 standard protocols, PRP and BC. PLT, WBC, and RBC counts, optical aggregometry assays, and PSL markers (pH, pCO2 , HCO3 , lactate and glucose concentrations, and LDH activity) were determined on storage days 0, 1, 3, 5, and 7. Aerobic and anaerobic bacterial cultures were also performed. RESULTS: Mean PLT counts were comparable between protocols and remained stable throughout storage up to day 7, while median WBC and RBC counts on day 0 were significantly higher in the BC-PC group (17,800 WBCs/µL; 195,000 RBCs/µL) than in the PRP-PC group (200 WBCs/µL; 10,000 RBCs/µL) (P = .012). In PRP-PC aggregometry, the median slope and amplitude in response to γ-thrombin and convulxin (+ ADP) were significantly decreased, and virtually absent in BC-PC during storage. PSL markers (lactate, LDH activity) were higher in BC-PC. Aerobic bacterial growth was observed in 2 PRP-PC and 1 BC-PC. CONCLUSIONS: This in vitro study suggests that PRP-PC had lesser WBC and RBC contamination and superior PLT function compared with BC-PC. In vivo studies are required to address safety and efficacy of PRP-PC.

Processing of equine bone marrow using the automated MarrowXpress System: RBC depletion, volume reduction, and mononuclear cell recovery.

BACKGROUND: The therapeutic use of bone marrow-derived mononuclear cells (MNCs) and mesenchymal stem cells for the treatment of soft tissue and orthopedic injuries in equine patients is expanding. After collection, bone marrow must be reduced in volume and depleted of RBCs for immediate therapeutic use or to prepare cells for culture or cryopreservation and storage. The MarrowXpress (MXP) System is an automated, closed, sterile system designed to process human bone marrow samples. OBJECTIVES: The purpose of this study was to evaluate the capacity of the MXP System to process equine bone marrow to reduce volume, deplete RBCs, and enhance recovery of MNCs. METHODS: Bone marrow was collected from 47 horses into 2 60-mL syringes containing heparin and processed using the MXP System. HCT, total nucleated cell (TNC) count, and MNC count were obtained for each sample before and after processing using an Advia 120 hematology analyzer. Volume reduction, RBC depletion, and recovery of TNCs and MNCs were calculated. RESULTS: For equine bone marrow samples, mean values were 73.2% for RBC depletion and 78.0% for volume reduction. TNC count before processing was 2.5 ± 1.2 × 10(7) and after processing was significantly higher at 7.8 ± 3.3 × 10(7) (P < .0001), with a recovery of 68.5 ± 24.5% (mean ± SD). MNC count before processing was 1.1 ± 0.9 × 10(7) and after processing was significantly higher at 3.8 ± 1.9 × 10(7) (P < .0001), with a recovery 73.0 ± 31.5%. CONCLUSIONS: The MXP System can reliably reduce volume and deplete RBCs from aspirates of equine bone marrow aspirates. MNCs can be recovered in a reproducible and sterile manner. Further studies evaluating the effects of the MXP System on cell viability, identification of mesenchymal stem cells (MSCs), and the efficacy of MSC expansion are warranted.