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  • Author or Editor: Guillermo Couto x
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in Journal of the American Veterinary Medical Association



To determine prevalence of p53 tumor suppressor protein overexpression in spontaneously arising tumors of dogs, using the CM-1 polyclonal antibody and immunohistochemical methods.

Design and Sample Population

Retrospective analysis was performed on archived, paraffin-embedded tumor tissue from dogs. A total of 226 tumors were evaluated, including tumors of epithelial, mesenchymal, and round cell origins.


Overexpression of p53 was detected by indirect immunohistochemical methods, using the CM-1 rabbit anti-human p53 polyclonal primary antibody. Protein overexpression was determined by use of a grading system based on percentage of stained tumor nuclei.


Nuclear overexpression of p53 was detected in most squamous cell carcinomas, nasal ade-nocarcinomas, and perianal gland adenocarcinomas. Hemangiopericytomas, transitional cell carcinomas, mammary adenocarcinomas, apocrine gland adenocarcinomas, intestinal adenocarcinomas, mast cell tumors, and cutaneous histiocytomas had low numbers of nuclei overexpressing p53. Remaining tumor types had intermediate p53 nuclear overexpression. Cytoplasmic staining was observed in some carcinomas, particularly intestinal adenocarcinomas.


Overexpression of p53 is common in spontaneously arising neoplasms of dogs.

Clinical Relevance

Prospective determination of p53 status in some tumor types may be as clinically useful in determining prognosis and predicting survival times for dogs with cancer as it is for human beings with cancer. (Am J Vet Res 1997;58:857–863)

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in American Journal of Veterinary Research


Objective—To evaluate the effects of various storage conditions on one-stage prothrombin time (OSPT), activated partial thromboplastin time (APTT), and fibrinogen concentration of canine plasma collected for transfusion.

Sample Population—Plasma from 9 dogs.

Procedure—Whole blood was collected from dogs by means of jugular venipuncture and centrifuged at 7,300 × g for 20 minutes at 0 C. A plasma extractor was then used to generate plasma. Aliquots of plasma were collected in segments of plastic tubing and in microcentrifuge tubes, and plasma collection bags, tubing segments, and microcentrifuge tubes were immediately frozen at –30 C. Additional tubing segments and microcentrifuge tubes were stored at 2 C. After 1 week of storage, all samples were thawed, and OSPT, APTT, and fibrinogen concentration were measured. Collection bags and microcentrifuge tubes were refrozen at –30 C, and values were measured again 30 days after blood collection.

Results—Values for OSPT, APTT, and fibrinogen concentration did not vary significantly with storage time, temperature, or container.

Conclusions and Clinical Relevance—Results suggested that storage for up to 30 days and at 2 C versus –30 C did not have any significant effect on hemostatic parameters of canine plasma obtained for transfusion. (Am J Vet Res 2001;62:734–735)

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in American Journal of Veterinary Research



To assess the effect of packed RBC (pRBC) transfusion on thromboelastographic (TEG) tracings in dogs with naturally occurring anemia.


22 clinically anemic dogs that received a pRBC transfusion.


For each dog, a blood sample was collected before and within 3 hours after completion of the pRBC transfusion for a CBC, nonactivated TEG analysis, and measurement of blood viscosity. Wilcoxon signed rank tests were used to compare CBC, viscosity, and TEG variables between pretransfusion and posttransfusion blood samples. Multivariable linear regression was used to assess the effects of pretransfusion-posttransfusion changes in Hct, WBC count, and platelet count on changes in TEG variables.


Median posttransfusion Hct (21%; range, 13% to 34%) was significantly greater than the median pretransfusion Hct (12.5%; range, 7% to 29%). Packed RBC transfusion was associated with a median increase in Hct of 6.2% (range, 1.2% to 13%). Maximum amplitude significantly decreased from 74.9 to 73.8 mm and clot strength significantly decreased from 14,906 to 14,119 dynes/s after pRBC transfusion. Blood viscosity significantly increased, whereas platelet and WBC counts significantly decreased after transfusion. Multivariable linear regression revealed that pretransfusion-posttransfusion changes in Hct, WBC count, and platelet count were not associated with changes in TEG variables.


Results indicated that pRBC transfusion had only small effects on the TEG tracings of hemodynamically stable dogs. Therefore, large changes in TEG tracings following pRBC transfusion are unlikely to be the result of the transfusion and should be investigated further.

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in American Journal of Veterinary Research



A 36-kg (79-lb) castrated male Greyhound (dog 1) and a 25-kg (55 lb) spayed female Greyhound (dog 2) underwent general anesthesia for dental care with similar perianesthetic protocols on multiple occasions from 2013 to 2016. Both dogs had periodontal disease but were otherwise deemed healthy. Both dogs developed clinically relevant hyperkalemia, with signs including loss of P waves on ECG tracings, during multiple anesthetic events.


Dog 1 developed hyperkalemia during 2 of 2 anesthetic events, with ECG changes noted during the first event. Dog 2 developed hyperkalemia during 3 of 4 anesthetic events, with ECG changes identified during the second and third events. Serum potassium concentration for both dogs was within the reference range prior to and between anesthetic events. No underlying etiopathogenesis for hyperkalemia was identified for either dog.


In each hyperkalemic event, the clinician stopped the dental procedure and continued to provide supportive care and monitoring while the dog recovered from anesthesia. The ECG changes resolved, and serum potassium concentration returned to the reference range rapidly after inhalant anesthetic administration was discontinued. The dogs were discharged from the hospital without further complications.


Hyperkalemia in anesthetized Greyhounds resulted in serious cardiac conduction abnormalities, which could be potentially fatal if not recognized and promptly treated. Further investigation into the etiopathogenesis, prevention and treatment strategies, and genetic or familial components of this condition is indicated.

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in Journal of the American Veterinary Medical Association


Chemotaxis under agarose was evaluated to establish an assay system and to characterize chemotactic responses of canine neutrophils. A method for the measurement of canine neutrophil chemotaxis was established, with optimal responses obtained with agarose containing 10% pooled canine serum, a concentration of 5 × 105 cells/well, zymosan-activated serum (zas), or autologous serum or plasma as the chemoattractants, and a 120-minute incubation period. Canine neutrophils responded well to zas, heat-inactivated zas, autologous serum and plasma, and heat-inactivated pooled serum. Chemotactic activity was proportional to the concentration of serum used as the chemoattractant. Mean (± sd) random migration, chemotaxis, chemotactic index, and chemotactic differential of neutrophils from 9 healthy Greyhounds were 1.09 (± 0.23), 1.95 (± 0.38), 1.82 (± 0.31), and 0.86 (± 0.32) mm, respectively.

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in American Journal of Veterinary Research


Objective—To evaluate the biological activity of dihydroartemisinin on canine osteosarcoma cell lines in vitro.

Sample Population—4 canine osteosarcoma cell lines.

Procedures—Cell viability assays were performed on canine osteosarcoma cell lines OSCA2, OSCA16, OSCA50, and D17 after 24, 48, and 72 hours of treatment with dihydroartemisinin at concentrations of 0.1 to 100μM. Apoptosis was assessed by use of an ELISA for free nuclosomal DNA fragmentation and by western blot analysis for cleavage of caspase 3. Cell cycle analysis was performed by use of staining with propidium iodide and flow cytometry. Detection of reactive oxygen species (ROS) was conducted in the D17 cell line by use of 6-carboxy-2′,7′-dihydrofluorescein diacetate and flow cytometry.

Results—The concentration of dihydroartemisinin required for 50% inhibition of cell viability (IC50) was achieved in all 4 canine osteosarcoma cell lines and ranged from 8.7 to 43.6μM. Induction of apoptosis was evident as an increase in nucleosomal DNA fragmentation, cleavage of caspase 3, and an increase in the population in the sub G0/G1 phase of the cell cycle detected by flow cytometry. Exposure to dihydroartemisinin also resulted in a decrease in the G0/G1 population. Iron-dependent generation of ROS was detected in dihydroartemisinin-treated D17 cells; ROS generation increased in a dose-dependent manner.

Conclusions and Clinical Relevance—Incubation with dihydroartemisinin resulted in biological activity against canine osteosarcoma cell lines, which included induction of apoptosis and arrest of the cell cycle. Clinical trials of dihydroartemisinin in dogs with osteosarcoma should be conducted.

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in American Journal of Veterinary Research


Objective—To compare WBC, neutrophil, and platelet counts and Hct values obtained with a point-of-care hematology analyzer with values obtained by a reference method for dogs and cats receiving chemotherapy.

Design—Cross-sectional study.

Animals—105 dogs and 25 cats undergoing chemotherapy.

Procedures—Blood samples were analyzed with a point-of-care hematology analyzer and with an impedance- and laser-based analyzer with manual differential WBC counts. Results for WBC, neutrophil, and platelet counts and Hct were compared. Sensitivity and specificity of the point-of-care analyzer to detect leukopenia, neutropenia, and anemia were calculated.

Results—554 canine and 96 feline blood samples were evaluated. Correlation coefficients for dogs and cats, respectively, were 0.92 and 0.95 for total WBC count, 0.91 and 0.88 for neutrophil count, 0.95 and 0.92 for Hct, and 0.93 and 0.71 for platelet count. Sensitivity and specificity, respectively, of the point-of-care analyzer to detect leukopenia were 100% and 75% for dogs and 100% and 68% for cats; to detect neutropenia were 80% and 97% for dogs and 100% and 80% for cats; to detect anemia were 100% and 80% for dogs and 100% and 66% for cats; and to detect thrombocytopenia were 86% and 95% for dogs and 50% and 87% for cats.

Conclusions and Clinical Relevance—The point-of-care analyzer was reliable for monitoring CBCs of dogs and cats receiving chemotherapy. It had good to excellent correlation for WBC and neutrophil counts and Hct and accurately detected leukopenia, neutropenia, and anemia. Sensitivity of the analyzer for detecting thrombocytopenia was lower but acceptable.

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in Journal of the American Veterinary Medical Association


Objective—To provide long-term follow-up information for a series of dogs and cats with invasive and noninvasive thymomas treated by excision alone.

Design—Retrospective case series.

Animals—9 cats and 11 dogs with thymoma.

Procedures—Medical records were reviewed. The following factors were analyzed for their effect on prognosis: age of dog or cat, invasiveness of the tumor, percentage of lymphocytes in the mass (percentage lymphocyte composition) on histologic evaluation, and mitotic index of the mass.

Results—All patients were treated with excision of the tumor alone. Median overall survival time for the cats was 1,825 days, with a 1-year survival rate of 89% and a 3-year survival rate of 74%. Median overall survival time for the dogs was 790 days, with a 1-year survival rate of 64% and a 3-year survival rate of 42%. Recurrence of thymoma was observed in 2 cats and 1 dog, and a second surgery was performed in each, with subsequent survival times of 5, 3, and 4 years following the first surgery. Percentage lymphocyte composition of the mass was the only factor that was significantly correlated with survival time; animals with a high percentage of lymphocytes lived longer.

Conclusions and Clinical Relevance—Results of this study indicated that most cats and dogs with thymomas did well after excision. Even cats and dogs with invasive masses that survived the surgery and the few cats and dogs with recurrent thymomas or paraneoplastic syndromes had a good long-term outcome. Excision should be considered an effective treatment option for dogs and cats with thymomas.

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in Journal of the American Veterinary Medical Association


OBJECTIVE To assess changes in biochemical and biophysical properties of canine RBCs during cold (1° to 6°C) storage in a licensed RBC additive solution (the RBC preservation solution designated AS-1) supplemented with ascorbic acid.

SAMPLE Blood samples from 7 neutered male Greyhounds; all dogs had negative results when tested for dog erythrocyte antigen 1.1.

PROCEDURES Blood was collected into citrate-phosphate-dextrose and stored in AS-1. Stored RBCs were supplemented with 7.1mM ascorbic acid or with saline (0.9% NaCl) solution (control samples). Several biochemical and biophysical properties of RBCs were measured, including percentage hemolysis, oxygen-hemoglobin equilibrium, and the kinetic rate constants for O2 dissociation, carbon monoxide association, and nitric oxide dioxygenation.

RESULTS Greyhound RBCs stored in AS-1 supplemented with ascorbic acid did not have significantly decreased hemolysis, compared with results for the control samples, during the storage period.

CONCLUSIONS AND CLINICAL RELEVANCE In this study, ascorbic acid did not reduce hemolysis during storage. Several changes in stored canine RBCs were identified as part of the hypothermic storage lesion.

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in American Journal of Veterinary Research