Studies on platelet aggregation using the Born method in normal and uraemic dogs

Using the Born method, based on light transmission in platelet rich plasma, the minimum effective concentration (threshold values) of several platelet agonists for inducing maximum platelet aggregation was determined in healthy dogs. The final concentrations of aggregation agonists were as follows: adenosine diphosphate (ADP) (0.5-50 micromol/L; n = 75 healthy dogs), collagen (0.5-20 mg/mL; n = 75), thrombin (0.1-5 IU/mL; n = 75), ristocetin (1-10 mg/mL; n = 10), and epinephrine (5-50 micromol/L; n = 10). Reference values for maximum aggregation with a lower limit of > 80% were achieved for agonist concentrations 25 micromol/L ADP (80-98%), > or = 10 microg/mL collagen (80-96%), and > or = 1 IU/mL thrombin (80-97%). None of the concentrations of epinephrine and ristocetin used in this study induced quantitative aggregation in the whole group of healthy dogs. We also studied platelet aggregation in 14 uraemic dogs using selected concentrations of aggregation agonists. Aggregation was significantly decreased in uraemic dogs using intermediate agonist concentrations, i.e., in the region of the threshold concentration. In contrast, maximum aggregation was increased in uraemic patients compared to reference values using low concentrations of all three agonists (ADP: 1 micromol/L, collagen: 1 microg/mL, and thrombin: 0.1, 0.2 IU/mL).     

Stage migration in dogs with lymphoma

BACKGROUND: Various diagnostic tests have been used to assign a clinical stage to dogs with lymphoma. As more sensitive staging methods are introduced, dogs are reclassified as having a higher disease stage, thereby affecting comparisons of dogs across differently staged clinical trials, and possibly, prognosis. HYPOTHESIS: The addition of more sensitive staging tests causes stage migration in dogs with lymphoma. ANIMALS: Fifty-nine client-owned dogs with previously untreated cytologically or histologically confirmed lymphoma METHODS: For every dog, the World Health Organization stage classification (I-V) was based on 5 groupings of various diagnostic tests: A (physical examination [PE] and quantitative blood count [QBC]), B (PE, QBC, thoracic and abdominal radiographs), C (PE, complete blood count with blood-smear evaluation [CBC], thoracic and abdominal radiographs), D (PE, CBC, thoracic radiographs, abdominal ultrasound), and E (PE, CBC, thoracic radiographs, abdominal ultrasound, and bone-marrow cytology). Dogs were treated with doxorubicin-based protocols. RESULTS: There was migration between all of the staging methods except D to E. However, the stage was not a predictor of remission rate, remission duration, or survival, regardless of staging method used. CONCLUSIONS AND CLINICAL IMPORTANCE: These data emphasized the need for standardized methods to determine the clinical stage in dogs with lymphoma.     

Whole blood platelet aggregation in dogs with liver disease

Whole blood platelet aggregation was determined in response to collagen, arachidonic acid, and adenosine diphosphate in 20 dogs with liver disease and in 20 control dogs. Platelet aggregation in response to collagen and arachidonic acid was reduced in dogs with liver disease, compared with control dogs (P less than 0.05), whereas there was no significant difference in platelet response to adenosine diphosphate between the 2 groups of dogs. Adenosine diphosphate was found not to be a reliable aggregation agent for determination of whole blood platelet aggregation in dogs. Dogs whose platelets did not aggregate in response to collagen and/or arachidonic acid manifested bleeding tendencies that could be attributed to platelet dysfunction.     

Whole blood platelet aggregation in uremic dogs

Whole blood platelet aggregation responses to collagen, arachidonic acid, and adenosine diphosphate were determined by use of the impedance method in 22 dogs with serum urea concentrations greater than or equal to 20 mmol/L, which was attributable to renal disease, and in 25 healthy control dogs. The median changes in impedance for the control dogs were 23 ohms for collagen, 18 ohms for arachidonic acid, and 6 ohms for adenosine diphosphate. The median changes in impedance in uremic dogs were 25 ohms for collagen, 21 ohms for arachidonic acid, and 15 ohms for adenosine diphosphate. There were no significant differences in platelet aggregation responses to collagen, arachidonic acid, and adenosine diphosphate between uremic and control dogs. Hemorrhagic tendencies were not detected in uremic dogs by use of whole blood platelet aggregation. Results of this study suggest that platelet aggregation by use of the whole blood platelet aggregometer is not abnormal in uremic dogs, but does not exclude the possibility of a platelet aggregation defect undetected by the whole blood system.     

Hematologic changes associated with half-body irradiation in dogs with lymphoma

BACKGROUND: Reports describe the technique and efficacy of half-body irradiation (HBI) of dogs with lymphoma, but few describe the distinctive toxicoses associated with the combination of HBI and chemotherapy. HYPOTHESIS: HBI would transiently affect myelocytic and erythroid variables as assessed by serial analysis of complete blood counts. ANIMALS: Twenty-nine dogs with lymphoma treated with HBI during 2002 and 2003. METHODS: A retrospective study of medical records of 29 dogs was performed. Two HBI protocols were used, resulting in delivery of either 6 Gy or 8 Gy to each half of the body, 1 month apart. Dogs received chemotherapy before, during, or after irradiation, or at multiple times. Serial hematology was available for all dogs. Data were analyzed between collection periods by analysis of variance (ANOVA) RESULTS: The mean granulocyte count significantly (P < .01) decreased from 10,017 cells/microL (data range 3,001-20,170 cells/ microL) before the first radiation treatment to 3,250 cells/microL (820-4,400 cells/microL) at week 5 (P < .01). Three weeks after this nadir, the mean increased to 10,150 cells/microL (900-26,700 cells/microL). The hematocrit did not change (36-38%). Thrombocytopenia (<100,000/microL) occurred in 10 dogs. Two dogs died because of complications associated with thrombocytopenia. No significant difference in toxicity was found between the 6 Gy and 8 Gy group. CONCLUSIONS AND CLINICAL IMPORTANCE: HBI was myelosuppressive but effects were short term and resolved in 22 of 24 dogs. Further studies are needed to elucidate the safety and role of HBI in the treatment of dogs with lymphoma.     

Evaluation of platelet aggregation using a point-of-care instrument in retired racing Greyhounds

Veterinarians involved in Greyhound rescue have anecdotally observed that 10-15% of Greyhounds bleed profusely after simple surgical procedures. In most patients, platelet counts and hemostasis profiles are normal; therefore, it is possible that these dogs have platelet dysfunction. The PFA-100 is a novel point-of-care platelet function analyzer that has recently been evaluated as a rapid method to assess platelet function in dogs. The objectives of this study were to characterize platelet function in a group of healthy Greyhounds by means of the PFA-100. Blood samples were collected from the jugular vein from 30 healthy Greyhounds. CBC, biochemical profile, PFA-100 assay with collagen/epinephrine (COL-EPI) and collagen/ adenosindiphosphate (COL-ADP), plasma von Willebrand factor antigen concentration (vWF:Ag), and vWF collagen-binding assay (vWF:CBA) were performed. PFA-100 closure times (CTs) with COL/ADP ranged from 63 to 92 seconds (mean +/- SD, 74.7 +/- 7.9 seconds) and with COL/EPI from 87 to 238 seconds (138 +/- 41 seconds); vWF: Ag ranged from 22 to 120% (87.52 +/- 25.5%) and vWF: CBA ranged from 36 to 102% (77.4 +/- 17.3%); and platelet counts ranged from 147 to 265 x 10(9)/L (194.6 +/- 31.64 x 10(9)/L). Greyhound CTs were significantly shorter than CTs in a mixed population of 50 healthy non-Greyhound dogs, in which the COL/ADP CTs ranged from 61 to 172 seconds (mean +/- SD, 87 +/- 21.6 seconds), and the COL/ EPI CTs ranged from 81 to 300 seconds (mean +/- SD, 183 +/- 67.6 seconds; P = 0.005 for COL/ADP CT; P = 0.001 for COL/ EPI CT). Also, platelet counts were significantly lower (P = 0.001) and packed cell volume was significantly higher (P = 0.001) in the Greyhound than in the non-Greyhound group. The PFA-100 is a reproducible method that can be used in the clinical setting to assess platelet function in Greyhounds; however, normal CTs in healthy Greyhounds are shorter than in other breeds. The results obtained in this study will be used to screen for abnormal platelet function in Greyhounds with postoperative bleeding.     

Platelet size,platelet surface-associated IgG,and reticulated platelets in dogs with immune-mediated thrombocytopenia

Abstract: Immune-mediated thrombocytopenia (IMT) is a disorder in which bound IgG on the surface of platelets results in platelet removal and alterations in mean platelet volume. Using flow cytometry, alterations in platelet size, platelet surface-associated IgG (PSAIgG), and numbers of reticulated platelets were determined in 13 dogs with primary IMT and 4 dogs with secondary IMT induced by experimental infection with Babesia gibsoni . Effects of sample age on platelet parameters also were determined, using samples from 20 dogs with normal platelet counts analyzed within 4 hours and after 24, 48, and 72 hours of storage in EDTA. No significant changes in platelet count, platelet size, or reticulated platelet percentage were observed in samples assayed within 4 and 24 hours of blood collection; whereas PSAIgG values increased 3 to 7 fold in samples stored for 24–72 hours. Using reference values for freshly collected or 24-hour-old samples, 10 of 13 (77%) dogs with primary IMT and all B gibsoni-inf ected dogs had increased PSAIgG levels. In 12 (75%) of the 16 dogs with thrombocytopenia the percentage of reticulated platelets was increased; however, absolute numbers of reticulated platelets were within reference values. Moreover, PSAIgG level and the percentage of reticulated platelets were not always increased concurrently in dogs with primary and secondary IMT. Platelet microparticles were detected in all B gibsoni-infected dogs, 8 of 13 (62%) dogs with primary IMT, and transiently in a dog that responded to immunosuppressive treatment. The results of this study indicate that sample age and time of sampling during disease affect interpretation of platelet parameters in dogs with IMT.     

Leukocyte and platelet changes following low-dose lipopolysaccharide administration in five dogs

Effects of low-dose LPS (0.1 μg/kg IV) on leukocyte and platelet parameters measured using an Advia 120 hematology analyzer were investigated. Five dogs received a saline sham treatment prior to LPS, and blood was collected before and 3, 6, and 24 h post-treatment. LPS-treated dogs had mild neutrophil toxic change and increased neutrophil bands at 3 and 6 h. Compared to saline-treated controls, total leukocyte, neutrophil, and monocyte counts of LPS-treated dogs were significantly decreased at 3 h and increased at 24 h. Compared to baseline, total leukocyte counts of LPS-treated dogs were significantly decreased at 3 h and increased at 24 h. Mean platelet volume was significantly increased and mean platelet component concentration was decreased at 3 h compared to baseline. Platelet count was significantly decreased at 3 and 6 h; plateletcrit did not change significantly. High dosage is not required in order to detect LPS-mediated hematologic effects in dogs. Low-dose LPS administration causes significant changes in leukocyte and platelet indices in dogs without causing severe clinical signs or death.     

Evaluation of the oral antimitotic agent (ABT-751) in dogs with lymphoma

Background

ABT‐751 is a novel orally available antimitotic agent that targets microtubule polymerization. This mechanism may suggest potential activity in canine lymphoma.

Objective

Determine a maximum tolerated dose for ABT‐751, and assess long‐term tolerability and activity in canine lymphoma.

Animals

Thirty dogs with newly diagnosed (n = 19) or relapsed (n = 11) non‐Hodgkin's lymphoma.

Methods

Dogs (n = 11) were enrolled in a rapid dose escalation study to define the maximum tolerated dose. Upon definition of a maximally tolerated dose, a cohort expansion of 19 dogs allowed verification of long‐term tolerability and assessment of activity. Study endpoints in the cohort expansion included chronic tolerability, response rate, response duration, and time to progression. Additional endpoints included serum pharmacokinetics, lymph node drug concentrations, and changes in circulating endothelial cells.

Results

The maximum tolerated dose of ABT‐751 was 350 mg/m² PO q24h. Dose‐limiting toxicities included vomiting and diarrhea, which resolved with a schedule adjustment to 350 mg/m² PO q48h. ABT‐751 was consistently detected in lymphoma tissue samples from dogs treated at or above the maximum tolerated dose. In the cohort expansion, objective responses were seen in 3/15 (20%) dogs with a response duration ranging from 21 to 111 days. Decreases in circulating endothelial cells were seen in 10 dogs at day 7 (2 responding dogs and 8 nonresponding dogs).

Conclusion

ABT‐751 was well tolerated at 350   mg/m² PO q24h for 7 days and then q48h thereafter. Activity of ABT‐751 suggested a rationale for additional studies of ABT‐751 as part of a combination chemotherapy protocol for lymphoma or other canine cancers.     

Comparison of COAP and UW-19 protocols for dogs with multicentric lymphoma

BACKGROUND: Various chemotherapy protocols for treating lymphoma in dogs have been published; however, comparison of protocols from different studies is difficult, especially when evaluating survival time and toxicoses. HYPOTHESIS: The choice of COAP (C, cyclophosphamide; O, vincristine; A, cytosine arabinoside; P, prednisone) and a modified University of Wisconsin 19-week (UW-19) induction protocol has no influence on overall survival times in dogs with lymphoma. ANIMALS: One hundred and one dogs with multicentric lymphoma. METHODS: Retrospective study (2001-2006). Dogs induced with either an 8-week COP-based protocol (C, cyclophosphamide; O, vincristine; and P, prednisone) with maintenance therapy (COAP group) or a 19-week CHOP (C, cyclophosphamide; H, doxorubicin; O, vincristine; and P, prednisone) based protocol (UW-19 group) were compared in terms of the duration of first remission, survival time, toxicoses, and cost. RESULTS: There were 71 dogs in the COAP group and 30 dogs in the UW-19 group. Various protocols were used after the first relapse. The median duration of the first remission for the COAP and UW-19 groups were 94 days (range, 6-356 days) and 174 days (28-438 days), respectively (P < .01). The median survival times for dogs in the COAP and UW-19 groups were 309 days (6-620 days) and 275 days (70-1102+ days), respectively (P = .09). Dogs in the COAP group had a hazard ratio of 1.9 (95% CI 1.1-3.4) for death relative to the UW-19 group (P = .03), after controlling for the confounders (World Health Organization clinical stage, age, sex, use of doxorubicin during reinduction). The severity of neutropenia and gastrointestinal toxicoses were significantly higher in the UW-19 group than in the COAP group (P = .01 and P < .01, respectively). CONCLUSION AND CLINICAL IMPORTANCE: Use of a long-term doxorubicin-containing sequential combination chemotherapy protocol is associated with a decreased risk of relapse and death relative to a non-doxorubicin-containing protocol.     

Comparison of 3 protocols for treatment after induction of remission in dogs with lymphoma

BACKGROUND: The optimal treatment after inducing complete remission (CR) in dogs with lymphoma has not been established. HYPOTHESIS: After inducing CR with L-asparaginase, vincristine, cyclophosphamide, doxorubicin, prednisone (L-CHOP); consolidation with either half-body radiation therapy (HBRT); or lomustine (CCNU) and mechlorethamine, vincristine, procarbazine, prednisone (MOPP) would improve first remission duration compared with continuing a CHOP-based protocol for an additional 4 months. ANIMALS: Dogs with stage III-V lymphoma. METHODS: Prospective clinical trial in which dogs initially were treated with an 8-week induction protocol that consisted of L-CHOP. Dogs in CR after induction were then allocated to 1 of 2 consolidation arms. A chemotherapy consolidation arm consisted of 2 treatments with CCNU and 1 cycle of MOPP. A HBRT arm consisted of 2 sequential 8.0-Gy fractions to the cranial and caudal half-body separated by 30 days. Vincristine was given between fractions. Results of the consolidation arms also were compared with a historical group treated with the same 8-week induction protocol followed by CHOP therapy until week 24. RESULTS: Overall, 67% of the dogs were in CR after 8 weeks of induction chemotherapy and were compared. Fifty-two dogs were in the historical arm, 23 in the CCNU/MOPP arm, and 27 in the HBRT arm. No difference in first remission duration was found among groups. Median first remission duration for the historical, CCNU/MOPP, and HBRT arms were 307, 274, and 209 days, respectively (P = .28). Overall second CR rate was 82% and was not different among groups (all P > or = .58). Overall remission duration (P = .28) and survival time (P = .48) were not different among groups. CONCLUSIONS AND CLINICAL IMPORTANCE: Consolidation with either CCNU/MOPP or HBRT showed no advantage over a standard CHOP-based protocol.     

Treatment of dogs with lymphoma using a 12-week, maintenance-free combination chemotherapy protocol

BACKGROUND: Treatment of lymphoma in dogs by long-term chemotherapy has favorable results. However, the efficacy of short-term, maintenance-free treatment protocols on remission and survival times in dogs has not been determined. HYPOTHESIS: That treatment using a 12-week chemotherapy protocol would be associated with satisfactory treatment outcome in dogs with lymphoma. ANIMALS: 77 dogs with histologically or cytologically confirmed diagnosis of lymphoma. METHODS: Prospective clinical trial in which dogs were treated with a 12-week chemotherapy protocol consisting of L-asparaginase, vincristine, cyclophosphamide, doxorubicin, and prednisolone. RESULTS: Complete remission rate was 76.3%. Multivariate logistic regression analysis revealed that clinical substage (P = .006) and immunophenotype (P = .003) had a significant influence on the likelihood of a dog achieving complete remission. Median duration of first complete remission was 243 days (range 19-1,191 days). The 6-month, 1-year, and 2-year remission rates were 68%, 28%, and 16%, respectively. In the multivariate analysis of patient variables, immunophenotype (P = .022) revealed a significant influence on first remission duration. Toxicosis was mild with the exception of 1 treatment-associated death. CONCLUSIONS AND CLINICAL IMPORTANCE: In this group of dogs the 12-week maintenance-free chemotherapy protocol was well tolerated and had satisfactory results.     

Evaluation of a 6-month chemotherapy protocol with no maintenance therapy for dogs with lymphoma

The purpose of this study was to compare a maintenance-free chemotherapy protocol based on CHOP (H from hydroxydaunorubicin = doxorubicin, O from Oncovin = vincristine) to a similar protocol with a maintenance phase for the treatment of canine lymphoma. Fifty-three dogs with multicentric lymphoma were treated with a 6-month modified version of the University of Wisconsin (UW)-Madison chemotherapy protocol (UW-25). Disease-free interval (DFI) and survival were compared to a historical control group of 55 dogs treated with a similar protocol with a prolonged maintenance phase. Remission rate for the study dogs was 94.2% (complete remission = 92.3%, partial remission = 1.9%). DFI and survival between the 2 groups did not differ significantly, with median DFI and survival of the study dogs equal to 282 and 397 days compared to 220 and 303 days for the control dogs (P = .2835 and .3365, respectively). Univariate analysis identified substage b (P = .0087), German Shepherd breed (P = .0199), and body weight > 18 kg (P = .0016) as significant for worse survival. Longer survival was associated with thrombocytopenia (P = .0436). Multivariate analysis revealed that substage (P = .0388) and weight (P = .0125) retained significance for DFI, whereas substage (P = .0093), thrombocytopenia (P = .0150), and weight (P = 0 .0050) retained significance for survival. Overall, the protocol was well tolerated by the dogs, with 41.5% (22/53) requiring a treatment delay or dose modification, but only 9.4% (5/53) needing hospitalization. The 6-month chemotherapy protocol based on CHOP with no maintenance phase provides similar DFI and survival times when compared to a similar protocol with a prolonged maintenance phase.     

Platelet function in dogs treated for lymphoma and hemangiosarcoma and supplemented with dietary n-3 fatty acids

A prospective randomized, double-blind clinical trial was performed to test the hypothesis that dogs with malignancies that are supplemented with n-3 fatty acids do not have clinical or laboratory evidence of coagulation disorders or altered platelet function when compared with unsupplemented dogs with similar malignancies. Thirteen dogs with hemangiosarcoma and 66 dogs with lymphoma were evaluated. Coagulation status of the dogs with lymphoma and hemangiosarcoma was evaluated with prothrombin time, partial thromboplastin time, platelet count, and in vitro platelet aggregometry using the whole-blood method. These tests were performed at 5 time points: before beginning the diet (week 0), at weeks 3, 15, and 21, and at 1 year or when progressive disease was evident. Alterations in platelet function in dogs receiving a diet supplemented with dietary n-3 fatty acids were not identified when compared to dogs fed a control diet. Dietary n-3 fatty acid supplementation using this dosage and ratio in dogs with lymphoma or hemangiosarcoma did not induce clinically significant hemorrhage in these animals. Therefore, supplementation with n-3 fatty acids did not result in clinical or laboratory evidence relating to uncontrolled hemorrhage in these dogs.     

Mushroom-derived maitake PETfraction as single agent for the treatment of lymphoma in dogs

BACKGROUND: Maitake PETfraction is a standardized essence extracted from the mushroom Maitake (Grifola frondosa) that has antitumor activity in tumor-bearing mice. In addition, PETfraction induces apoptosis in human prostate and bladder cancer cells and suppresses the proliferation in vitro of several canine tumor cell lines, such as lymphoma (Cl-1), mammary gland (CF33), and connective tissue (CF21). HYPOTHESIS: Maitake PETfraction is effective as a single agent in dogs with lymphoma. ANIMALS: Fifteen dogs with confirmed intermediate or high-grade lymphoma were enrolled into this prospective, noncontrolled, clinical trial. Inclusion criteria were an expected survival time of at least 2 weeks and no major organ dysfunction. METHODS: Maitake PETfraction was administered at a dose of 3 drops/kg/day divided into 2 doses given 1 hour before feeding. Dogs were evaluated by physical examination with tumor measurement, body weight, CBC, and chemistry profile before treatment and after 2, 4, 8, and 12 weeks. At each visit, owners completed a questionnaire addressing overall quality of life, appetite, and any adverse effects noted. RESULTS: A decrease in lymph node size of greater than 50% (objective response) was not seen in any of the dogs. Thirteen dogs developed progressive disease before the 4th week. The median treatment duration was 27 days (range, 9-228). PETfraction was well accepted, and minimal adverse effects were observed. Two dogs developed hyphema. It was not known if this was related to progressive lymphoma or was an adverse effect of treatment. CONCLUSIONS: No objective responses were observed to administration of Maitake PETfraction, and the drug was well tolerated in these dogs.     

Platelet aggregation in dogs with mitral valve regurgitation

OBJECTIVE: To compare platelet aggregation in healthy dogs and dogs with mitral valve regurgitation (MVR) to determine whether regurgitation had an effect on platelet function. ANIMALS: 32 dogs with MVR and 43 healthy dogs. PROCEDURE: Platelet aggregation was measured with an aggregometer, using adenosine 5'-diphosphate as the aggregating agent, and the maximum aggregation and the enhancement of platelet sensitivity (EPS) values were calculated. RESULTS: Platelet count and maximum aggregation were not significantly different between healthy dogs and dogs with MVR. However, EPS values in dogs with MVR were significantly higher than values in healthy dogs. Platelet count and maximum aggregation were not significantly different between dogs classified as New York Heart Association functional class I or II and dogs classified as functional class III or IV; however, EPS values were significantly higher in dogs classified as functional class III or IV. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that platelet aggregation is decreased in dogs with MVR and that the EPS value may be more sensitive to differences in disease severity than in measurement of maximum aggregation.