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Abstract

Objective—To examine clinical features, laboratory test results, treatment, and outcome of dogs with pure red cell aplasia (PRCA) and idiopathic nonregenerative immune-mediated anemia (NRIMA).

Design—Retrospective study.

Animals—43 dogs with severe nonregenerative anemia.

Procedure—Medical records of dogs determined to have PRCA, NRIMA, or ineffective erythropoiesis on the basis of bone marrow analysis between 1988 and 1999 were reviewed. Criteria for inclusion were ≥ 5- day history of severe nonregenerative anemia (Hct < 20%; < 60.0 X 103 reticulocytes/µl) with no underlying diseases. Information was retrieved on signalment, clinical signs, laboratory test results, treatment, and outcome.

Results—Median age of the dogs was 6.5 years. Spayed females and Labrador Retrievers were significantly overrepresented. Median Hct was 11% with no evidence of regeneration (median, 1.5 X 103 reticulocytes/ µl). Direct Coombs' test results were positive in 57% of dogs. Biochemical abnormalities included hyperferremia and high percentage saturation of transferrin. Bone marrow findings ranged from PRCA (5%) to erythroid hyperplasia (55%). Myelofibrosis was common. Dogs were treated with immunosuppressive drugs and the response was complete, partial, and poor in 55, 18, and 27% of the dogs, respectively. Mortality rate was 28%.

Conclusion and Clinical Relevance—An immunemediated pathogenesis should be considered in dogs with severe, nonregenerative anemia, normal WBC and platelet counts, hyperferremia, mild clinical signs, and no evidence of underlying disease. Bone marrow findings range from the rare PRCA to erythroid hyperplasia. Myelofibrosis is often detected in affected dogs and may prevent bone marrow aspiration. (J Am Vet Med Assoc 2000;216:1429–1436)

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in Journal of the American Veterinary Medical Association

Abstract

Objective—To determine the effect of citrate concentration (3.2 vs 3.8%) on coagulation tests in dogs.

Design—Original study.

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)

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in Journal of the American Veterinary Medical Association

Abstract

OBJECTIVE To measure thrombin generation by high and low tissue factor (TF)–expressing canine cancer cell lines.

SAMPLE Canine cell lines CMT25 (high TF–expressing mammary gland tumor cell line) and HMPOS (low TF–expressing osteosarcoma cell line).

PROCEDURES Thrombin generation by cancer cells was measured in pooled normal canine plasma by use of calibrated automated thrombography without added trigger reagents. Results were expressed as lag time, time to peak thrombin concentration, peak thrombin concentration, and total thrombin concentration or thrombin generation potential. Corn trypsin inhibitor, hirudin, and annexin V were used to inhibit contact activation, thrombin formation, and phosphatidylserine activity, respectively. Pooled normal human plasma deficient in coagulation factors VII, VIII, IX, X, XI, or XII was used to assess the role of individual coagulation factors on thrombin generation.

RESULTS CMT25 generated significantly more thrombin than did HMPOS (mean ± SD, 3,555 ± 604nM thrombin•min and 636 ± 440nM thrombin•min, respectively). Thrombin generation of CMT25 was dependent on factor VII and phosphatidylserine and was independent of contact activation. In contrast, thrombin generation of HMPOS was attributed to contact activation.

CONCLUSIONS AND CLINICAL RELEVANCE High TF-expressing canine mammary cancer cells generated thrombin in a plasma milieu in vitro in a factor VII- and phosphatidylserine-dependent manner. These findings support a role for TF in hypercoagulability detected in dogs with mammary gland tumors and potentially for other tumors that strongly express TF.

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in American Journal of Veterinary Research

Abstract

OBJECTIVE To evaluate expression of procoagulant tissue factor (TF) by canine hemangiosarcoma cells in vitro.

SAMPLES 4 canine hemangiosarcoma cell lines (SB-HSA [mouse-passaged cutaneous tumor], Emma [primary metastatic brain tumor], and Frog and Dal-1 [primary splenic tumors]) and 1 nonneoplastic canine endothelial cell line (CnAoEC).

PROCEDURES TF mRNA and TF antigen expression were evaluated by quantitative real-time PCR assay and flow cytometry, respectively. Thrombin generation was measured in canine plasma and in coagulation factor–replete or specific coagulation factor–deficient human plasma by calibrated automated thrombography. Corn trypsin inhibitor and annexin V were used to examine contributions of contact activation and membrane-bound phosphatidylserine, respectively, to thrombin generation.

RESULTS All cell lines expressed TF mRNA and antigen, with significantly greater expression of both products in SB-HSA and Emma cells than in CnAoEC. A greater percentage of SB-HSA cells expressed TF antigen, compared with other hemangiosarcoma cell lines. All hemangiosarcoma cell lines generated significantly more thrombin than did CnAoEC in canine or factor-replete human plasma. Thrombin generation induced by SB-HSA cells was significantly lower in factor VII–deficient plasma than in factor-replete plasma and was abolished in factor X–deficient plasma; residual thrombin generation in factor VII–deficient plasma was abolished by incubation of cells with annexin V. Thrombin generation by SB-HSA cells was unaffected by the addition of corn trypsin inhibitor.

CONCLUSIONS AND CLINICAL RELEVANCE Hemangiosarcoma cell lines expressed procoagulant TF in vitro. Further research is needed to determine whether TF can be used as a biomarker for hemostatic dysfunction in dogs with hemangiosarcoma.

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in American Journal of Veterinary Research

Abstract

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.

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in American Journal of Veterinary Research

Abstract

Objective—To determine whether canine tumor cell lines express functional tissue factor and shed tissue factor-containing microparticles.

Sample—Cell lines derived from tumors of the canine mammary gland (CMT12 and CMT25), pancreas (P404), lung (BACA), prostate gland (Ace-1), bone (HMPOS, D-17, and OS2.4), and soft tissue (A72); from normal canine renal epithelium (MDCK); and from a malignant human mammary tumor (MDA-MB-231).

Procedures—Tissue factor mRNA and antigen expression were evaluated in cells by use of canine-specific primers in a reverse transcriptase PCR assay and a rabbit polyclonal anti-human tissue factor antibody in flow cytometric and immunofluorescent microscopic assays, respectively. Tissue factor procoagulant activity on cell surfaces, in whole cell lysates, and in microparticle pellets was measured by use of an activated factor X-dependent chromogenic assay.

Results—Canine tissue factor mRNA was identified in all canine tumor cells. All canine tumor cells expressed intracellular tissue factor; however, the HMPOS and D-17 osteosarcoma cells lacked surface tissue factor expression and activity. The highest tissue factor expression and activity were observed in canine mammary tumor cells and pulmonary carcinoma cells (BACA). These 3 tumors also shed tissue factor-bearing microparticles into tissue culture supernatants.

Conclusions and Clinical Relevance—Tissue factor was constitutively highly expressed in canine tumor cell lines, particularly those derived from epithelial tumors. Because tumor-associated tissue factor can promote tumor growth and metastasis in human patients, high tissue factor expression could affect the in vivo biological behavior of these tumors in dogs.

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in American Journal of Veterinary Research

Abstract

Objective—To use a chromogenic assay to measure tissue factor (TF) activity on the cell surface and in whole cell lysates of feline monocytes in response to treatment with lipopolysaccharide (LPS) and fetal bovine serum (FBS).

Animals—14 healthy cats.

Procedures—Peripheral blood monocytes were isolated via density gradient centrifugation followed by adhesion to plastic. Tissue factor procoagulant activity was measured by use of an assay that detects TF-activated factor X, on the basis of cleavage of a chromogenic TF-activated factor X–dependent substrate. Activity was quantified by comparison with a serially diluted human recombinant TF-activated factor × curve.

Results—The TF procoagulant activity assay was sensitive and specific for TF. Treatment with LPS stimulated TF procoagulant activity on the surface and in whole cell lysates of isolated feline leukocytes. The LPS response in intact cells was dose dependent and cell number dependent and was inhibited by FBS. Monocyte isolation was inefficient, with monocytes comprising a mean of 22% of the isolated cells.

Conclusions and Clinical Relevance—A TF-activated factor X–dependent chromogenic assay that uses human reagents successfully measured surface-expressed and intracellular TF activity of feline monocytes. Treatment with LPS induced TF expression on feline monocytes, but this response was inhibited by FBS. The chromogenic assay was a useful method for measuring TF procoagulant activity in feline cells in vitro and can be used as a research tool to investigate the role of cell-associated TF in thrombotic disorders in cats.

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in American Journal of Veterinary Research

Abstract

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)

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in American Journal of Veterinary Research

Abstract

CASE DESCRIPTION

A 17-year-old Friesian gelding was examined at a referral hospital because of a 1-month history of mild exercise intolerance and marked lymphocytosis.

CLINICAL FINDINGS

Physical examination revealed no peripheral lymphadenopathy or other abnormalities. Results of an abdominal palpation examination per rectum and thoracic and abdominal ultrasonographic examinations were unremarkable. B-cell chronic lymphocytic leukemia (CLL) was diagnosed on the basis of severe lymphocytosis and positive expression of the B-cell marker CD20 by lymphocytes in the bone marrow and peripheral blood.

TREATMENT AND OUTCOME

Treatment with prednisolone (2 mg/kg [0.9 mg/lb], PO, every other day) and chlorambucil (20 mg/m2, PO, every 3 weeks for 2 doses, then every 2 weeks) was initially associated with improvement in clinical signs and a decrease in the lymphocyte count. However, 3 weeks after administration of the first dose of chlorambucil, the lymphocyte count began to increase. One week later, the horse developed episodes of recurrent fever and the lymphocyte count continued to increase. Despite continued administration of the prednisolone-chlorambucil protocol, the horse's clinical condition deteriorated rapidly, and it was euthanized 6 weeks after initial examination at the referral hospital because of a poor prognosis. A necropsy was not performed.

CLINICAL RELEVANCE

B-cell CLL has been infrequently described in horses. This report was the first to describe the use of chemotherapy, albeit unsuccessful, for the treatment of B-cell CLL in a horse. This information should be useful for guiding expectations for prognosis and management of other horses affected with the disease.

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in Journal of the American Veterinary Medical Association