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Case Description—A 23-year-old Thoroughbred mare was evaluated because of a coagulopathy causing hemoperitoneum, hematomas, and signs of blood loss–induced anemia.
Clinical Findings—The mare had tachycardia, pallor, hypoperfusion, and a large mass in the right flank. The mass was further characterized ultrasonographically as an extensive hematoma in the body wall with associated hemoabdomen. Coagulation testing revealed persistent, specific prolongation of the activated partial thromboplastin time (> 100 seconds; reference interval, 24 to 44 seconds) attributable to severe factor VIII deficiency (12%; reference interval, 50% to 200%). On the basis of the horse's age, lack of previous signs of a bleeding diathesis, and subsequent quantification of plasma factor VIII inhibitory activity (Bethesda assay titer, 2.7 Bethesda units/mL), acquired hemophilia A was diagnosed. The medical history did not reveal risk factors or underlying diseases; thus, the development of inhibitory antibodies against factor VIII was considered to be idiopathic.
Treatment and Outcome—The mare was treated with 2 transfusions of fresh whole blood and fresh-frozen plasma. Immunosuppressive treatment consisting of dexamethasone and azathioprine was initiated. Factor VIII deficiency and signs of coagulopathy resolved, and the inhibitory antibody titer decreased. The mare remained healthy with no relapse for at least 1 year after treatment.
Conclusions and Clinical Relevance—Horses may develop inhibitory antibodies against factor VIII that cause acquired hemophilia A. A treatment strategy combining transfusions of whole blood and fresh-frozen plasma and administration of immunosuppressive agents was effective and induced sustained remission for at least 1 year in the mare described here.
Objective—To measure platelet membrane–derived microparticle (PMP) content and thrombin-generating capacity of canine plasma subjected to specific processing and storage conditions.
Animals—31 clinically normal dogs (19 males and 12 females).
Procedures—Citrate-anticoagulated blood samples obtained from each dog were centrifuged at 2,500 × g to isolate platelet-poor plasma (PPP), then PPP was centrifuged at 21,000 × g to isolate microparticle-free plasma (MPF) and microparticle-enriched plasma (MPEP). Whole blood and paired samples of fresh and frozen-thawed PPP, MPF, and MPEP were dual labeled for flow cytometric detection of membrane CD61 (constitutive platelet antigen) and annexin V (indicating phosphatidylserine externalization). Platelets and PMPs were enumerated with fluorescent, size-calibrated beads. Thrombin generation in fresh and frozen-thawed PPP, MPF, and MPEP was measured via kinetic fluorometric assays configured with low tissue factor and low phospholipid concentrations.
Results—Initial centrifugation yielded PPP with < 0.5% the platelets of whole blood, with median counts of 413 PMPs/μL for males and 711 PMPs/μL for females. Sequential centrifugation resulted in a 10-fold concentration of PMPs in MPEP and virtually depleted PMPs from MPF. Thrombin generation depended on PMP content, with median endogenous thrombin potential of 0, 893, and 3,650 nmol•min for MPF, PPP, and MPEP, respectively. Freeze-thaw cycling caused significant increases in PMP counts and phosphatidylserine externalization.
Conclusions and Clinical Relevance—Canine PMPs were major determinants of thrombin-generating capacity; preanalytic variables influenced plasma PMP content. Processing conditions described here may provide a basis for characterization of PMPs in clinical studies of thrombosis in dogs.
Objective—To determine the effect of citrate concentration (3.2 vs 3.8%) on coagulation tests in dogs.
Animals—30 clinically healthy dogs and 12 dogs with hereditary hemostatic disorders.
Procedure—Blood was collected from all dogs directly into collection tubes containing 3.2 or 3.8% buffered citrate. Prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen concentration were measured by use of 3 clot-detection assay systems (2 mechanical and 1 photo-optic). Factor VIII and factor IX coagulant activities (FVIII:C and FIX:C, respectively) were determined by use of a manual tilt-tube method and a mechanical clot-detection device.
Results—Significant differences were not detected in median PT, fibrinogen concentration, FVIII:C, or FIX:C between 3.2 and 3.8% citrate for any assay system. A significant prolongation in aPTT for 3.2% citrate, compared with 3.8% citrate, was found in 1 mechanical system.
Conclusions and Clinical Relevance—Citrate concentration does not significantly affect results of most coagulation assays, regardless of assay system. The aPTT was mildly influenced by the citrate concentration, although this was animal-, instrument-, and reagent-dependent. The choice of 3.2 or 3.8% citrate as an anticoagulant for coagulation tests has minimal influence on assay results in healthy dogs or dogs with hereditary hemostatic disorders. (J Am Vet Med Assoc 2000;217:1672–1677)
Objective—To identify hemostatic imbalances indicative of an increased risk of intra-abdominal adhesion formation in foals versus adult horses.
Animals—Horses with colic undergoing exploratory laparotomy or abdominocentesis as part of a clinical examination (n = 16 foals ≤ 6 months of age and 19 adults ≥ 5 years of age) and horses without colic undergoing herniorrhaphy (15 foals) or euthanasia for noninflammatory and nongastrointestinal disease (10 foals and 20 adults).
Procedures—Paired abdominal fluid and blood samples were collected from each horse into buffered sodium citrate and centrifuged immediately after collection. Supernatants were stored at −80°C, then thawed for measurement of fibrinogen concentration, plasminogen activity, antiplasmin activity, and D-dimer concentration. Supernatant analyte concentrations or activities were compared within age group (foals with and without colic vs adults with and without colic) and within disease status (foals and adults without colic vs foals and adults with colic).
Results—All analyte concentrations or activities in abdominal fluid samples were significantly lower in the noncolic groups than in the colic groups, and none differed between foal and adult groups. Several plasma analyte values differed by disease status and age.
Conclusions and Clinical Relevance—The risk of intra-abdominal adhesion formation in the foals in this study did not appear to be attributable to differences in intra-abdominal hemostasis between adult horses and foals. Strategies for initial medical and surgical management of colic in adult horses may be applicable to foals with similar disorders.
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 determine sensitivity and specificity of assays of D-dimer concentrations in dogs with disseminated intravascular coagulation (DIC) and healthy dogs and to compare these results with those of serum and plasma fibrin-fibrinogen degradation product (FDP) assays.
Animals—20 dogs with DIC and 30 healthy dogs.
Procedure—Semi-quantitative and quantitative D-dimer concentrations were determined by use of latex-agglutination and immunoturbidometry, respectively. Fibrin-fibrinogen degradation products were measured by use of latex-agglutination. A reference range for the immunoturbidometric D-dimer concentration assay was established; sensitivity and specificity of the assay were determined at 2 cutoff concentrations (0.30 µg/ml and 0.39 µg/ml).
Results—Reference range for the immunoturbidometric D-dimer concentration assay was 0.08 to 0.39 µg/ml; median concentrations were significantly higher in dogs with DIC than in healthy dogs. Latexagglutination D-dimer and serum and plasma FDP assays had similar sensitivity (85 to 100%) and specificity (90 to 100%); the immunoturbidometric assay had lower specificity (77%) at the 0.30 µg/ml cutoff and lower sensitivity (65%) at the 0.39 µg/ml cutoff. Sensitivity or specificity of the latex-agglutination D-dimer assay was not significantly improved when interpreted in series or parallel with FDP assays.
Conclusions and Clinical Relevance—Measurement of D-dimer concentrations by latex-agglutination appears to be a sensitive and specific ancillary test for DIC in dogs. Specificity of D-dimer concentrations in dogs with systemic disease other than DIC has not been determined, therefore FDP and D-dimer assays should be performed concurrently as supportive tests for the diagnosis of DIC in dogs. (Am J Vet Res 2000;61:393–398)
Objective—To determine whether dogs with naturally occurring canine parvoviral (CPV) enteritis have laboratory evidence of hypercoagulability.
Animals—9 dogs with naturally occurring CPV enteritis and 9 age-matched control dogs.
Procedure—Blood was collected from all dogs within 24 hours of admission for thromboelastography (TEG) and determination of activated partial thromboplastin time (aPTT), prothrombin time (PT), antithrombin III (AT) activity, and fibrinogen concentration. Fibrin-fibrinogen degradation product (FDP) concentration, Ddimer concentration, and platelet count were obtained in dogs with CPV enteritis only. Records were reviewed for evidence of thrombosis or phlebitis.
Results—All 9 dogs with CPV enteritis had evidence of hypercoagulability, determined on the basis of significantly increased TEG maximum amplitude and decreased AT activity. Fibrinogen concentration was significantly higher in dogs with CPV enteritis than in control dogs. The aPTT was moderately prolonged in dogs with CPV enteritis, and FDP concentration was < 5 mg/ml in 7 of 9 dogs. No dogs had a measurable D-dimer concentration. Platelet counts were within reference range. Four of 9 dogs had clinical evidence of venous thrombosis or phlebitis associated with catheters. One dog had multifocal splenic thrombosis identified at necropsy.
Conclusions and Clinical Relevance—Dogs with CPV enteritis have a high prevalence of clinical thrombosis or phlebitis and laboratory evidence of hypercoagulability without disseminated intravascular coagulopathy. Thromboelastography may help identify hypercoagulable states in dogs. (J Am Vet Med Assoc 2000;217:1500–1504)
Objective—To define the relationship between clinical expression of a type-1 von Willebrand disease phenotype and genotype at 2 von Willebrand factor marker loci in Doberman Pinschers.
Animals—102 client-owned Doberman Pinschers.
Procedures—Dogs were recruited on the basis of plasma von Willebrand factor concentration, clinical history, and pedigree. Blood samples and response to a history questionnaire were obtained for each dog. Plasma von Willebrand factor concentration was measured by use of an ELISA, and genotyping was performed via polymerase chain reaction for 1 intragenic and 1 extragenic von Willebrand factor marker. Amplification product size was determined by use of polyacrylamide gel electrophoresis (intragenic marker) or automated sequence analysis (extragenic marker). Western blots were prepared from a subset of dogs with low plasma von Willebrand factor concentration to evaluate multimer distribution.
Results—Strong associations were detected between plasma von Willebrand factor concentration and von Willebrand factor marker genotype. Twentyfive dogs had substantial reduction in plasma von Willebrand factor concentration and multiple hemorrhagic events. All were homozygous for a 157-basepair intragenic marker allele and homozygous or compound heterozygous for 1 of 4 extragenic marker alleles. These marker genotypes were exclusively detected in dogs with low plasma von Willebrand factor concentration, although some dogs with these genotypes did not have abnormal bleeding.
Conclusions and Clinical Relevance—Type-1 von Willebrand disease in Doberman Pinschers is associated with the von Willebrand factor gene locus; however, the expression pattern in this breed appears more complex than that of a simple recessive trait. (Am J Vet Res 2001;62:364–369)
Objective—To evaluate the clinical course of dogs with hemophilia A (factor VIII deficiency) and to determine whether factor VIII coagulant activity (FVIII:C) was associated with severity of clinical signs and outcome.
Sample—Respondent information for 39 client-owned dogs with FVIII deficiency.
Procedures—Information was obtained via a survey distributed to the American College of Veterinary Internal Medicine and American College of Veterinary Emergency and Critical Care email list serves and to the Veterinary Information Network community to identify dogs with hemophilia A (FVIII:C ≤ 20%). Severity of FVIII deficiency was classified as mild (FVIII:C, 6% to 20%), moderate (FVIII:C, 2% to 5%), or severe (FVIII:C, < 2%).
Results—Data for 39 dogs (38 males and 1 female) were compiled. Mixed-breed dogs, German Shepherd Dogs, and Labrador Retrievers were most commonly affected. In most (34/39) dogs, disease was diagnosed at < 1 year of age. Bleeding associated with teething, minor trauma, vaccination, and elective surgical procedures most commonly prompted FVIII:C testing. Affected dogs had similar signs of spontaneous hemorrhage regardless of the magnitude of FVIII deficiency. Four dogs were euthanized without treatment at the time of diagnosis. Thirty dogs received ≥ 1 blood transfusion; FVIII:C did not appear to influence transfusion requirements.
Conclusions and Clinical Relevance—Results indicated that dogs with hemophilia A have variations in clinical course of the disease and may have a good long-term prognosis. Residual FVIII:C may not be useful for predicting severity of clinical signs, transfusion needs, or long-term prognosis.
Objective—To evaluate the platelet activation response before and after treatment with clopidogrel in horses.
Animals—12 healthy adult mares.
Procedures—In a masked study, horses (6/group) were randomly allocated to alternately receive placebo or clopidogrel via nasogastric tube at a loading dose of 4 mg/kg followed by 2 mg/kg every 24 hours. Blood samples were collected before and 72 hours after initiation of treatment for ADP- and collagen-induced light transmission aggregometry; determination of closure time in collagen-ADP cartridges; modified thrombelastography for comparison of maximal amplitudes generated by kaolin, reptilase, and reptilase plus ADP activation; and flow cytometric tests to detect platelet fibrinogen binding, P-selectin expression, and phosphatidylserine externalization before and after ex vivo stimulation with thrombin, convulxin, thrombin with convulxin, and calcium ionophore.
Results—Clopidogrel administration induced a significant decrease in mean aggregation response to 5μM and 10μM ADP stimulation; however, 2 horses had resistance to clopidogrel's inhibitory action. Significant differences after clopidogrel treatment were not found in any other tests of platelet function.
Conclusions and Clinical Relevance—Assays using commercially available reagents were configured to measure different variables of the platelet activation response; however, clopidogrel's platelet inhibitory action was only detected by ADP-induced light transmission aggregometry. Results also suggested that horses, like humans, have interindividual variability in response to clopidogrel that may influence the drug's clinical efficacy as an antiplatelet agent.