Investigation of platelet measurands in dogs with hematologic neoplasia.

INTRODUCTION: Thrombocytopenia has been associated with some neoplastic processes, including hematologic neoplasia. There is no information regarding specific changes in platelet measurands in dogs with hematologic neoplasia compared with healthy dogs. The objectives of our study were to establish RIs, evaluate platelet measurands in dogs with hematologic neoplasia, and compare these measurands in patients with hematologic malignancies with or without thrombocytopenia. METHODS: This was a retrospective study. Platelet measurands were determined using the ADVIA 120 Hematology analyzer when a CBC was performed and included the platelet count, MPV, platelet distribution width (PDW), plateletcrit (PCT), mean platelet component (MPC), platelet component distribution width (PCDW), mean platelet mass (MPM), platelet mass distribution width (PMDW), and number of large platelets. Reference intervals were determined retrospectively using data from 129 healthy dogs. Patients with hematologic neoplasia (n = 50) were identified through retrospective evaluation of medical records from the Auburn University Veterinary Teaching Hospital and separated into thrombocytopenic (n = 20) and nonthrombocytopenic groups (n = 30). RESULTS: Platelet count and PCT were significantly higher in older healthy dogs compared with younger dogs. Significant differences were identified when comparing healthy dogs with those with hematologic neoplasia without thrombocytopenia for PDW, PCDW, PMDW, and the number of large platelets, indicating the presence of more heterogeneous platelets. Thrombocytopenic dogs with hematologic neoplasia had significantly decreased MPCs and increased MPVs, MPMs, and PCDWs compared with nonthrombocytopenic dogs with neoplasia. CONCLUSIONS: Dogs with hematologic neoplasia had more heterogeneous platelets, whereas thrombocytopenic patients with neoplasia had more activated platelets.

Effect of lactic acid addition to equine whole blood on platelet aggregation measured by impedance aggregometry.

BACKGROUND: Acidemia in sick or injured horses is often due to lactic acid accumulation. Alterations in platelet function and hemostasis are among numerous deleterious effects caused by decreased physiologic pH. OBJECTIVES: We aimed to evaluate the effect of hyperlactatemia and resultant acidemia on platelet aggregation in equine whole blood using impedance aggregometry. METHODS: Platelet aggregation was measured using the Multiplate analyzer in whole blood from 34 healthy horses at baseline and after in vitro addition of lactic acid to adjust the pH. Platelet aggregation of each sample was quantified by the area under the curve measurement reported by the Multiplate system. The association between platelet aggregation and pH was analyzed using a linear mixed-effects model. The association of baseline platelet aggregation with hematocrits (Hcts), white blood cell (WBC) counts, and platelet counts was evaluated using Pearson's correlations. RESULTS: There was a significant association between acidemia and decreased platelet aggregation. No significant correlations were detected between platelet aggregation and Hct, WBC count, or platelet count. Platelet aggregation measured in healthy horses using the Multiplate analyzer showed substantial variation between animals. CONCLUSIONS: Acidemia caused by the addition of lactic acid to equine whole blood was associated with a mild though statistically significant decrease in platelet aggregation. In conjunction with other factors, this change may contribute to morbidity-related disorders of hemostasis, although its precise clinical relevance is uncertain.

Cancer of unknown primary in a mare: case report and comparative pathology review.

A 25-y-old Percheron mare was admitted to the teaching hospital because of lethargy and intractable dyspnea. Thoracoabdominal ultrasound examination identified severe peritoneal effusion, mild bilateral pleural effusion, and a diffuse pulmonary nodular pattern. Cytology of peritoneal fluid revealed a hypercellular sample with clusters of neoplastic polygonal cells and admixed macrophages. Euthanasia was followed by postmortem examination; marked bi-cavitary effusion was present, and innumerable up to 4-cm diameter, round-to-floriform nodules were diffusely evident throughout serosal surfaces as well as the pulmonary and hepatic parenchyma. Disseminated adenocarcinoma, predominantly affecting lung and liver with widespread serosal implantation, was confirmed on light microscopy. Neoplastic cells had strong immunolabeling for pancytokeratin and lacked immunoreactivity to vimentin, napsin A, and Pax8. Cytokeratin 7 and thyroid transcription factor-1 were non-contributory given absent and inconsistent internal control reactivity, respectively. Such results, combined with the lack of a major mass that would indicate a primary site, were supportive of carcinoma of unknown primary site, which remains a conundrum in human oncology, and is poorly explored in veterinary medicine, mainly as a result of clinical and diagnostic limitations.

Clinical Feasibility and Airway Deposition of Nebulized Voriconazole in Healthy Horses.

Voriconazole (VRC) is a potential treatment for pneumomycosis in horses. The objectives of this study were to determine if the delivery of Vfend using a Flexineb nebulizer produced clinically significant [VRC] in lower airways. The hypothesis was that [VRC] after delivery by nebulization would be greater in the pulmonary epithelial lining fluid than plasma. A secondary objective was to determine [VRC] in upper airways through the collection of nasopharyngeal wash (NPW) samples. Voriconazole solution [Vfend-6.25 mg/mL, 100 (n = 2), 200 (n = 3), 500 (n = 1) mg] was nebulized once in 6 healthy geldings. Clinical responses, duration of nebulization, and [VRC] at various time points (up to 8 hours) in plasma, bronchoalveolar lavage fluid (BALF) supernatant and cell pellet, and NPW samples were recorded. Voriconazole (Vfend-6.25 mg/mL, 200 mg) was nebulized in 5 additional, healthy geldings, and [VRC] was measured in NPW samples pre- and postnebulization at time points up to 8 hours. The antifungal activity of BALF and NPW samples was determined using agar disk diffusion. Concentrations of voriconazole were below detection in plasma, BALF supernatant, and cell pellets for all time points and doses except the BALF cell pellet (0.4 µg/g) immediately after nebulization of 500 mg. For 5 horses, administered 200 mg of Vfend, mean [VCR] in NPW at the end of nebulization and 1, 6, and 8 hours postnebulization were: 30.8 ± 29, 1.0 ± 0.84, 0.2 ± 0.19, and 0.34 ± 0.67 µg/mL, respectively. Only NPW samples obtained immediately postnebulization showed antifungal activity. A nebulized Vfend solution is not recommended for the treatment of pneumomycosis in horses.

The effect of adding lactic acid to canine whole blood on platelet aggregation as measured by impedance aggregometry.

BACKGROUND: Acidemia in sick dogs often results from the accumulation of lactic acid. The resulting decrease in blood pH can have many physiologic effects, including alteration of platelet function. OBJECTIVES: We aimed to evaluate the effect of hyperlactatemia and subsequent acidemia on platelet aggregation in canine blood using impedance aggregometry. METHODS: Platelet aggregation was measured in blood from 27 healthy dogs using the Multiplate analyzer at baseline and after in vitro addition of two different volumes of lactic acid to adjust the pH. The area under the curve (AUC), reported by the Multiplate analyzer, was used to assess the extent of platelet aggregation in each sample. A linear mixed effects model was used to test for the association between platelet aggregation and pH. The association of baseline platelet aggregation with HCTs, platelet counts, and WBC counts was assessed using Pearson's correlations. RESULTS: Acidemia was associated with a significant decrease in platelet aggregation. No significant correlations were detected between platelet aggregation and HCT, platelet count, or WBC count. Platelet aggregation measured using the Multiplate analyzer showed substantial individual variation. CONCLUSIONS: Worsening acidemia due to the addition of lactic acid caused a mild but significant decrease in platelet aggregation in canine blood. The clinical significance of this change is uncertain but could be important when combined with other abnormalities of hemostasis associated with illness.

Retrospective evaluation of serum/plasma iron, red blood cell distribution width, and nucleated red blood cells in dogs with acute trauma (2009-2015): 129 cases.

OBJECTIVE: To compare the prognostic value of admission hematologic parameters serum/plasma iron, red blood cell distribution width (RDW), and nucleated red blood cells (nRBCs) in dogs presenting with acute traumatic injury. DESIGN: Retrospective observational study (2009-2015). SETTING: University teaching hospital. ANIMALS: One hundred and twenty-nine clinical dogs presenting within 24 hours of acute traumatic injury. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: One hundred and twenty-nine dogs met the inclusion criteria and 109 (84.5%) survived, while 20 (15.5%) died or were euthanized in hospital. Patients with blunt force trauma comprised 79.8% of the patient population; dogs with penetrating trauma comprised 20.2% of cases. Hypoferremia occurred in all nonsurvivors, and the median serum/plasma iron concentration was significantly lower in nonsurvivors than survivors (P = 0.028). Normal or increased serum/plasma iron had 100% specificity and 100% positive predictive value for survival. Red blood cell distribution width was not significantly different between groups (P = 0.417). The presence of nRBCs was significantly associated with nonsurvival (P = 0.030), although the absolute nRBC concentrations were not significantly different (P = 0.070). A multiple logistic regression model found age, type of injury, presence of nRBCs, and serum/plasma iron to be independent predictors of survival with an area under the receiver operator characteristic curve of 0.813. CONCLUSIONS: The presence of nRBCs and low serum/plasma iron are associated with mortality in patients with acute trauma; however, red blood cell distribution width was not associated with survival. Absence of hypoferremia was highly associated with a favorable prognosis in this patient population. These parameters may warrant inclusion in trauma scoring systems.

Evaluation of Histogel- and Gelfoam-embedded bronchoalveolar lavage and transtracheal wash fluids compared with cytocentrifuged and sediment smear preparations.

BACKGROUND: Storage and temperature significantly impact bronchoalveolar lavage fluid (BALF) analysis, and shipment of samples to diagnostic laboratories is often necessary. Alternative sample preparation methods could limit storage and temperature effects. OBJECTIVES: This study aimed to determine if airway wash samples that were fixed in formalin after being embedded in Histogel or Gelfoam gave comparable results to fresh cytocentrifuged or sediment smear preparations for the evaluation of cell morphology. METHODS: Eleven bronchoalveolar lavage and 3 transtracheal wash fluids were available, including 8 canine, 1 feline, and 5 equine samples. Cytocentrifuged and sediment smear preparations were prepared for routine analysis. Airway fluids were reserved for further evaluation. Total nucleated cell counts (TNCCs) were determined using a hemocytometer. The remaining fluid was used for Histogel and Gelfoam preparations. Each preparation was analyzed by a single board-certified clinical pathologist and assigned cellularity (1-3) and morphology scores (1-4). RESULTS: Cellularity and morphology were significantly worse for the sediment smear, Histogel, and Gelfoam preparations compared with the cytocentrifuged preparations. The Gelfoam preparations had significantly worse cellularity scores than all other methods. Cellularity scores for sediment smears and Histogel preparations were significantly correlated with TNCCs. CONCLUSIONS: TNCCs impacted the cellularity of the sediment smears and Histogel preparations. Cytocentrifuged preparations resulted in the best cellularity and morphology and are, therefore, recommended whenever possible. Neither the Histogel nor the Gelfoam methods demonstrated any advantage over sediment smear preparations, and both performed poorly when compared with cytocentrifuged preparations. Therefore, we do not recommend the use of these methods.

In vitro effect of blood cell counts on multiple-electrode impedance aggregometry in dogs.

OBJECTIVE To assess the effect of decreased platelet and WBC counts on platelet aggregation as measured by a multiple-electrode impedance aggregometer in dogs. ANIMALS 24 healthy dogs. PROCEDURES From each dog, 9 mL of blood was collected into a 10-mL syringe that contained 1 mL of 4% sodium citrate solution to yield a 10-mL sample with a 1:9 citrate-to-blood ratio. Each sample was then divided into unmanipulated and manipulated aliquots with progressively depleted buffy-coat fractions such that 2 to 3 blood samples were evaluated per dog. The Hct for manipulated aliquots was adjusted with autologous plasma so that it was within 2% of the Hct for the unmanipulated aliquot for each dog. All samples were analyzed in duplicate with a multiple-electrode impedance aggregometer following the addition of ADP as a platelet agonist. The respective effects of platelet count, plateletcrit, Hct, and WBC count on platelet aggregation area under the curve (AUC), aggregation, and velocity were analyzed with linear mixed models. RESULTS WBC count was positively associated with platelet AUC, aggregation, and velocity; blood samples with leukopenia had a lower AUC, aggregation, and velocity than samples with WBC counts within the reference range. Platelet count, plateletcrit, and Hct did not have an independent effect on AUC, aggregation, or velocity. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that WBC count was positively associated with platelet aggregation when ADP was used to activate canine blood samples for impedance aggregometry. That finding may be clinically relevant and needs to be confirmed by in vivo studies.

A novel form of macrothrombocytopenia in Akita dogs.

Blood samples from 3 unrelated Akita dogs with a common history of persistent macrothrombocytopenia in the absence of clinical bleeding were sent to the Auburn University College of Veterinary Medicine (AUCVM) Clinical Pathology Laboratory for evaluation. Due to low platelet counts, one Akita dog had been treated with corticosteroids for presumed immune-mediated platelet destruction, and one Akita dog was treated with doxycycline for one month for presumed infection by a tick-borne agent. In spite of treatment, platelet counts remained low in both dogs. Given the absence of abnormal bleeding in all 3 dogs and lack of response to treatment in 2, congenital macrothrombocytopenia was suspected. Interestingly, platelets from all 3 dogs exhibited a consistent elongated platelet morphology. There were no morphologic abnormalities observed in other cell lines. While there have been anecdotal reports of a possible inherited macrothrombocytopenia in Akita dogs, scientific studies have not been done to verify these reports. This manuscript represents the first case report describing what is likely a congenital macrothrombocytopenia in Akita dogs based on persistently low platelet counts in the absence of clinical signs, and characterized by a unique platelet morphology.

Hematologic findings predictive of bone marrow disease in dogs with multicentric large-cell lymphoma.

BACKGROUND: Evaluation of the bone marrow is needed for complete staging in dogs with multicentric large-cell lymphoma, but is often omitted in clinical practice. OBJECTIVES: The objective was to determine if routine peripheral blood findings, including microscopic evaluation of blood smears, can predict the presence of bone marrow involvement in dogs with lymphoma. METHODS: Hematologic data including evaluation of blood smears and bone marrow aspirates from 107 dogs newly diagnosed with large-cell lymphoma were retrospectively evaluated. Neoplastic lymphocytes were identified based on cell size, nuclear size, chromatin pattern, and the presence of nucleoli. Positive specimens were defined as having ≥ 10% neoplastic lymphocytes. Two groups were established based on the presence or absence of lymphoma in the bone marrow. Variables (positive blood smear, HCT, platelet count, and total and differential WBC counts) were evaluated to determine if they were predictive of bone marrow involvement using univariate and multivariate logistic models. RESULTS: Thrombocytopenia and the presence of > 10% neoplastic lymphocytes on blood smears were identified as significant variables for predicting the presence of bone marrow involvement. When considered independently, either a positive blood smear or thrombocytopenia had low sensitivity (60%) and moderate specificity (89% and 87%, respectively). Sensitivity increased when these variables were evaluated together (80%). CONCLUSIONS: In dogs with multicentric large-cell lymphoma, thrombocytopenia or the presence of neoplastic lymphocytes in circulation is suggestive of bone marrow involvement, but not definitive. Normal peripheral blood findings do not exclude the possibility of lymphoma in the bone marrow.

Multifocal cutaneous histiocytic sarcoma in a young dog and review of histiocytic cell immunophenotyping.

A 9-month-old male Great Dane had progressive generalized nodular dermatopathy for several months. There were > 100 raised, alopecic, firm, painful nodules throughout the skin. Aspirates from several lesions yielded moderate numbers of irregularly round or polygonal to spindle-shaped cells with mild to moderate anisocytosis and few inflammatory cells, and the cytologic interpretation was proliferation of mesenchymal or histiocytic cells. On histopathologic examination, nodules were composed of densely packed sheets of round to spindle-shaped cells with mild anisokaryosis and low mitotic activity. Multifocal histiocytic sarcoma with a spindle-cell pattern was diagnosed based on morphologic features and intense expression of CD18. Additional immunophenotypic analysis on frozen sections of tissue confirmed the diagnosis of histiocytic sarcoma; expression of CD18, CD45, CD1a, CD11b, and CD11c, limited expression of Thy-1 (CD90) and CD80, and lack of expression of CD4, CD11d, and CD86 indicated that the cells were likely interstitial dendritic cells; a review of reactive and neoplastic dendritic cells is provided. Based on staging, internal organs were not affected. Sequential treatment with lomustine and doxorubicin failed to prevent progression of the cutaneous lesions, and the dog died 3 months after initial diagnosis. At necropsy, a focus of neoplastic cells was present in one lymph node, but except for skin other organs were not involved. The clinical presentation of histiocytic sarcoma may be unusual, and neoplastic cells may lack overt features of malignancy on cytologic and histopathologic examination. In some instances, immunophenotyping is required to differentiate histiocytic sarcoma from other histiocytic disorders.

Evaluation of HistoGel™-embedded specimens for use in veterinary diagnostic pathology.

HistoGel™ is an aqueous specimen-processing gel that encapsulates and suspends histologic and cytologic specimens in a solidified medium. HistoGel-embedded specimens can then be processed and evaluated by routine histologic and immunohistochemical methods. This methodology has been used in human diagnostic pathology and is especially useful for small, friable, or viscous tissue samples that are difficult to process. In addition, special histochemical stains or immunohistochemistry can be performed on HistoGel-embedded cytologic specimens using standardized methods developed for histopathology. The current report describes several applications for HistoGel, including use with cytologic specimens, bone marrow aspirates, retention of tissue orientation for endoscopic biopsy specimens, and evaluation of friable tissues. Samples were encapsulated in HistoGel, fixed in 10% neutral buffered formalin, routinely processed, paraffin embedded, and sectioned for histochemical and immunohistochemical evaluation. The results of this study support the use of HistoGel in veterinary diagnostic pathology.

Evaluation and clinical application of platelet function testing in small animal practice.

Tests that evaluate many aspects of platelet function have been applied in both human and veterinary medicine for the monitoring of treatment with platelet function inhibitors and for detection of platelet function abnormalities (inherited or acquired). Interspecies variation in the response to various platelet agonists is an important consideration when methods that have been developed for people are applied in other species. At the present time, many of these assays are not readily available in standard veterinary practice. Advanced platelet function testing for veterinary patients is offered at select academic institutions. Discussion with a specialist is recommended when considering the use of these tests, and the relative strengths and limitations of each assay should be considered in the interpretation of test results.

Resistance of canine lymphoma cells to adenoviral infection due to reduced cell surface RGD binding integrins.

Recombinant adenovirus vectors (Ad) have been recognized as effective in vivo gene delivery vehicles and utilized as gene therapy agents for a number of cancers. The elucidation of viral entry mechanisms has allowed the development of recombinant vectors that exploit existing cell surface receptors to achieve entry into the cell. B lymphocytes are normally resistant to infection by adenovirus 5, likely due to the lack of the Coxsackie and Adenovirus receptor (CAR). Using reverse-transcriptase PCR and flow cytometry, the CD40 receptor has been shown to be expressed on many lymphoma cells. We exploited this finding to develop a gene therapy strategy for treatment of canine B cell lymphoma. Ad5 was targeted to cells expressing CD40 via CD40 ligand (CD40L) and was effective in infecting CD40-expressing control cells; however, both primary canine lymphoma cells and cell lines demonstrated limited evidence of transduction. Following receptor binding, adenovirus entry into cells may require interaction with α(v)ß(3/5) integrins; we demonstrate that canine lymphoma cells are deficient in these integrins. Reduced α(v)ß(3) integrin expression may render these cells incapable of internalizing Ad vectors. Thus, any viral targeting approaches for treatment of canine lymphoma must also take into account the potential lack of internalization signals.

Diagnostic accuracy of gray-scale ultrasonography for the detection of hepatic and splenic lymphoma in dogs.

Gray-scale ultrasonography is often used to screen for involvement of the liver and spleen in canine lymphoma patients but the utility of sonography for staging lymphoma has not been evaluated quantitatively. We performed abdominal sonography in 28 dogs with a confirmed diagnosis of lymphoma. Needle aspirates were obtained for cytology from three separate sites in the liver and three sites in the spleen and the sonographic appearance was noted at each site. Our hypothesis was that in dogs newly diagnosed with lymphoma, abnormal appearance of the liver or spleen on ultrasound examination is an indication that lymphoma is present in that organ. Cytologic evaluation was used as the gold standard. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of hepatic and splenic abnormalities seen on ultrasound for diagnosis of lymphoma were 72.7%, 80.6%, 77.4%, 76.3%, and 76.8% for the liver, respectively, and 100%, 23.3%, 64.6%, 100%, and 68.1% for the spleen, respectively. Based on these results, we recommend that aspirates be performed for detection of lymphoma in the spleen of dogs only when the spleen appears abnormal ultrasonographically and that cytology of the liver be performed, regardless of ultrasonographic appearance, to determine the presence or absence of lymphoma.

Unique structure of the M loop region of ß1-tubulin may contribute to size variability of platelets in the family Felidae.

BACKGROUND: Platelet size is relatively uniform in mammals except for domestic cats. Uniform platelet production by megakaryocytes can be disrupted if microtubule assembly or dynamics is impaired. Mutations in the gene encoding ß1-tubulin have been documented in dogs and people, and the resulting microtubule effects have been associated with production of large platelets. OBJECTIVES: The objectives of this study were to evaluate morphology of platelets on feline blood smears, determine the gene sequences encoding ß1-tubulin in members of the family Felidae, and compare the findings with those in other mammalian species to determine whether predicted structural differences in ß1-tubulin that might affect microtubule stability or assembly were present. METHODS: At least 100 platelets/smear on blood smears from 15 domestic cats and 88 big cats were evaluated to assess platelet size variability. Platelet-derived cDNA obtained from a domestic cat and genomic DNA isolated from blood samples of domestic cats and other members of the family Felidae were analyzed by PCR using primers specific for ß1-tubulin. Gene sequences obtained were compared with those of other common mammals. RESULTS: Two differences in gene sequence were found in a highly conserved region encoding the M loop of ß1-tubulin in members of the family Felidae compared with sequences from other species. Platelet size variation was present in big cats and domestic cats. In addition, a rare amino acid change was documented in the C-terminal region encoding the H11 helix in domestic cats. CONCLUSION: Members of the family Felidae have an altered M loop region in ß1-tubulin compared with other mammals. This variation may contribute to the observed platelet size variability.

Use of infrared spectroscopy for diagnosis of traumatic arthritis in horses.

OBJECTIVE: To evaluate use of infrared spectroscopy for diagnosis of traumatic arthritis in horses. ANIMALS: 48 horses with traumatic arthritis and 5 clinically and radiographically normal horses. PROCEDURES: Synovial fluid samples were collected from 77 joints in 48 horses with traumatic arthritis. Paired samples (affected and control joints) from 29 horses and independent samples from an affected (n = 12) or control (7) joint from 19 horses were collected for model calibration. A second set of 20 normal validation samples was collected from 5 clinically and radiographically normal horses. Fourier transform infrared spectra of synovial fluids were acquired and manipulated, and data from affected joints were compared with controls to identify spectroscopic features that differed significantly between groups. A classification model that used linear discriminant analysis was developed. Performance of the model was determined by use of the 2 validation datasets. RESULTS: A classification model based on 3 infrared regions classified spectra from the calibration dataset with overall accuracy of 97% (sensitivity, 93%; specificity, 100%). The model, with cost-adjusted prior probabilities of 0.60:0.40, yielded overall accuracy of 89% (sensitivity, 83%; specificity, 100%) for the first validation sample dataset and 100% correct classification of the second set of independent normal control joints. CONCLUSIONS AND CLINICAL RELEVANCE: The infrared spectroscopic patterns of fluid from joints with traumatic arthritis differed significantly from the corresponding patterns for controls. These alterations in absorption patterns may be used via an appropriate classification algorithm to differentiate the spectra of affected joints from those of controls.