Determination of clopidogrel effect in cats using point-of-care Plateletworks ADP and shipped samples for PFA-200 analysis in a clinical practice setting.

OBJECTIVES: Clopidogrel is the recommended first-line antithrombotic in cats for a variety of conditions; however, it is ineffective in 15-20% of cats. The determination of clopidogrel effectiveness with platelet function assays has historically been limited to specialty centers; however, recent work has suggested that in-hospital or shipped analyses of samples may be feasible. The aim of the present study was to investigate the utility of an in-house analysis and shipping of blood samples collected in primary practices for the determination of clopidogrel effectiveness. METHODS: Citrated blood samples were collected from cats receiving clopidogrel therapy by veterinarians in clinical practices across Canada, a median of 304.4 km from the reference laboratory (range 8-4425). Samples were analyzed in-house using Plateletworks ADP and shipped for remote analysis using PFA-200 P2Y and COL/ADP cartridges. RESULTS: A total of 30 samples were collected from 25 cats. Of these, the percentage of samples analyzable for the presence or absence of the clopidogrel effect was 86% for Plateletworks ADP, 90% for PFA-200 P2Y and 87% for PFA-200 COL/ADP. There was no significant difference in the number of samples unable to be analyzed by each modality (P = 0.689) due to flow obstruction or other sample characteristics. The prevalence of absence of clopidogrel effectiveness on platelet function assays was 8% with the PFA-200 COL/ADP assay, 25% with the PFA-200 P2Y assay and 30% with the Plateletworks ADP assay. CONCLUSIONS AND RELEVANCE: The results of this study confirm that samples of feline blood can be collected in clinical practices and shipped to a reference laboratory for PFA-200 analysis with a high rate of success, comparable to point-of-care analysis.

Effects of Atorvastatin and Rosuvastatin on Blood Lipids in Quaker Parrots (Myiopsitta monachus).

Statin drugs are the most effective class of hypolipidemic and antiatherosclerotic drugs, with atorvastatin and rosuvastatin being the most effective. While the use of statins would be a tremendous asset in the treatment of dyslipidemia and lipid-accumulation disorders in birds, there are only limited data available regarding their use and effectiveness in psittacine species. Two consecutive randomized crossover trials on Quaker parrots (Myiopsitta monachus) were performed to study the effect of atorvastatin and rosuvastatin. Ten birds were used in an initial balanced crossover experiment with 5 oral treatments (control; atorvastatin 10 mg/kg q12h and q24h; rosuvastatin 10 mg/kg q12h and q24h) for 2 weeks each. Plasma lipidomics and lipoprotein profiling were performed after each treatment. Twelve birds were used in a second experiment consisting of 2 parallel crossover studies, each with 6 birds either fed their regular diet or a 0.3% cholesterol diet. In the 2 parallel crossover studies, the treatment group was administered atorvastatin 20 mg/kg orally q12h and the control group a placebo suspension orally q12h. Plasma lipidomics, lipoprotein profiles, and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity were subsequently measured. Results were analyzed with serial linear mixed models and trends were assessed graphically. No statistically significant effect of any statin treatment was detected on plasma lipids, lipoproteins, creatinine kinase, or HMG-CoA reductase activity. In the first trial, all the rosuvastatin treatments led to some nonsignificant decreases in several triacylglycerol species, while in the second trial this was only observed in the birds on atorvastatin 20 mg/kg q12h being fed their regular diet. Quaker parrots may require much higher doses of statin drugs to show significant and clinically useful lipid-lowering effects.

Validation of storage and shipping of feline blood samples for analysis on the Platelet Function Analyzer-200 for determining the effect of clopidogrel.

BACKGROUND: The Platelet function analyzer-200 (PFA-200) can determine the effect of clopidogrel in cats, but analysis traditionally must be performed at point-of-care (POC). The ability to ship samples of blood to a laboratory would allow widespread access. OBJECTIVES: We aimed to validate the shipping of blood samples for PFA-200 analysis in cats to determine the effect of clopidogrel. METHODS: Twenty healthy cats and 10 cats receiving clopidogrel were recruited. Blood was collected from cats and aliquoted into two samples, one was analyzed at POC within 2 hours using the PFA-200, and the other was packaged and transported to a location 4 km away, stored, and transported back to the lab for analysis the following day. RESULTS: Median closure times (CTs) with the collagen/adenosine diphosphate (COL/ADP) cartridge in healthy cats were 51.5 seconds (POC) and 78.8 seconds (shipped), which were significantly different (P < 0.001), and for cats on clopidogrel, median CTs were 147.5 seconds (POC) and 190 seconds (shipped), which were not significantly different (P = 0.131). Median CTs with the P2Y cartridge in healthy cats were 50.5 seconds (POC) and 64.9 seconds (shipped), which were significantly different (P = 0.03), and in cats receiving clopidogrel, median CTs were 300 seconds (POC) and 300 seconds (shipped) which were not significantly different (P = 1.000). Reference intervals for CTs differed for COL/ADP at POC (19.8-89.7 seconds) and shipped (50.9-161.6 seconds) and for P2Y at POC (35.5-118.8 seconds) and shipped (35.1-108.9 seconds). Receiver operating characteristics showed similar areas under the curve (AUCROCs) regarding the effect of clopidogrel for COL/ADP at POC (0.994 seconds) and shipped (0.932) and for P2Y at POC (0.904 seconds) and shipped (0.975 seconds). When classifying for the presence of clopidogrel effects, Cohen's Kappa was 0.62 for COL/ADP and 1.00 for P2Y. CONCLUSIONS: Shipping blood samples for PFA analysis are feasible with similar performance to POC analyses for determining the effect of clopidogrel in cats.

Treatment of myeloid neoplasia with doxorubicin and cytarabine in 11 dogs.

Myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) are primary myeloid neoplasms in dogs generally considered to have a poor outcome. In this study, we assessed toxicity, efficacy and outcome of concurrent administration of doxorubicin and cytarabine in 11 dogs with myeloid neoplasia. Bone marrow specimens were reviewed by three pathologists and classified as either MDS (n = 2), high grade MDS/early AML (MDS/AML; n = 4) or AML (n = 5). The median number of treatment cycles was 5 (range 1-9) and resolution of cytopenia was reported in 7 of 11 dogs including 2 dogs with MDS, 2 dogs with MDS/AML, and 3 dogs with AML. The median duration of remission in the seven responders was 344 days (range 109-1428) and the median overall survival for all dogs was 369 days. Adverse events consisted of predominantly low-grade gastrointestinal illness and myelosuppression. Three dogs developed grade V toxicity manifesting with heart failure (n = 2) at 369 and 1170 days after diagnosis and acute gastrointestinal side effects (n =1). Despite a limited sample size, these results suggest that a doxorubicin and cytarabine protocol may be considered as a therapeutic option in dogs with myeloid neoplasia. Protocol safety, in particular regarding myocardial toxicity, and efficacy should be further investigated.

Neonatal hyperleukocytosis and regenerative anemia in a septic puppy.

This paper reports a case of neonatal hyperleukocytosis in a dog due to a bacterial infection. A 3-week-old, mixed-breed dog was brought to a veterinary college referral center with a history of weight loss despite a good appetite. Clinical and laboratory examinations included: physical examination, complete blood (cell) count (CBC), serum biochemistry profile, abdominal ultrasound examination, and cytology of liver and bone marrow aspirates. The CBC showed hyperleukocytosis of 158.0 × 109/L (RI: 2.1 to 21.2 × 109/L) and hematocrit of 0.19 L/L (RI: 0.21 to 0.34 L/L). The strong leukemoid reaction was comprised of neutrophils, monocytes, and lymphocytes. The dog was diagnosed with Staphylococcus pseudointermedius liver infection based on liver aspirates and culture. Amoxicillin-clavulanic acid was prescribed. A recheck abdominal ultrasound and CBC repeated 4 wk after initial examination were unremarkable. Neonatal hyperleukocytosis is well-described in human medicine but veterinary studies in small animal neonates are scarce. Key clinical message: Hyperleukocytosis in adult dogs may be caused by leukemia or leukemoid reactions. Generalized sepsis is a leading cause of leukemoid reactions in adult dogs and cats. In puppies, neoplasia is less likely, and other causes should be investigated. Similar to human neonates, puppies can mount a strong leukemoid reaction during an infection, even if it is not a generalized septic process.

Hyperleucocytose néonatale et anémie régénérative chez un chiot septique. Cet article rapporte un cas d'hyperleucocytose néonatale chez un chien dû à une infection bactérienne. Un chien de race mixte âgé de 3 semaines a été amené dans un centre de référence d'une école vétérinaire avec des antécédents de perte de poids malgré un bon appétit. Les examens cliniques et de laboratoire comprenaient : examen physique, numération globulaire complète (CBC), profil biochimique sérique, examen échographique abdominal et cytologie des aspirations du foie et de la moelle osseuse.Le CBC montrait une hyperleucocytose de 158,0 × 109/L (RI : 2,1 à 21,2 × 109/L) et un hématocrite de 0,19 L/L (RI : 0,21 à 0,34 L/L). La forte réaction leucémique était composée de neutrophiles, de monocytes et de lymphocytes. Le chien a été diagnostiqué avec une infection hépatique à Staphylococcus pseudointermedius sur la base d'aspirations et de cultures de foie. L'amoxicilline-acide clavulanique a été prescrit. Une échographie abdominale de contrôle et un CBC répété 4 semaines après l'examen initial étaient sans particularité. L'hyperleucocytose néonatale est bien décrite en médecine humaine mais les études vétérinaires chez les nouveau-nés de petits animaux sont rares.Message clinique clé :L'hyperleucocytose chez les chiens adultes peut être causée par une leucémie ou des réactions leucémiques. La septicémie généralisée est l'une des principales causes de réactions leucémiques chez les chiens et les chats adultes. Chez les chiots, la néoplasie est moins probable et d'autres causes doivent être recherchées. Semblables aux nouveaunés humains, les chiots peuvent développer une forte réaction leucémique lors d'une infection, même s'il ne s'agit pas d'un processus septique généralisé.(Traduit par Dr Serge Messier).

Evaluation of lymphocyte-specific programmed cell death protein 1 receptor expression and cytokines in blood and urine in canine urothelial carcinoma patients.

Urothelial carcinoma (UC) is the most common urinary tumour in dogs. Despite a range of treatment options, prognosis remains poor in dogs. In people, breakthroughs with checkpoint inhibitors have established new standards of care for muscle-invasive bladder cancer patients and elevated levels of programmed cell death protein 1 (PD-1) suggest immune checkpoint blockade may be a novel target for therapy. The goal of this study was to determine if canine UC patients express elevated levels of lymphocyte-specific PD-1 and/or urinary cytokine biomarkers compared to healthy dogs. Paired blood and urine were evaluated in 10 canine UC patients, five cystitis patients and 10 control dogs for lymphocyte-specific PD-1 expression via flow cytometry and relative cytokine expression. In UC patients, PD-1 expression was significantly elevated on CD8+ lymphocytes in urine samples. UC patients had a higher CD4:CD8 ratio in their urine compared to healthy dogs, however, there was no significant variation in the CD8:Treg ratio between any group. Cystitis patients had significantly elevated levels of CD4+ T cells, CD8+ T cells and Tregs in their blood samples compared to UC patients and healthy dogs. Cytokine analysis demonstrated significant elevations in urinary cytokines (granulocyte-macrophage colony-stimulating factor, interferon-gamma [IFN-γ], interleukin (IL)-2, IL-6 IL-7, IL-8 and IL-15, IP-10, KC-like, IL-18, monocyte chemoattractant protein-1 and tumour necrosis factor-alpha). Several of these cytokines have been previously correlated with both lymphocyte-specific PD-1 expression (IFN-γ, IL-2, IL-7 and IL-15) in muscle-invasive urothelial carcinoma in humans. Our results provide evidence of urinary lymphocyte PD-1 expression and future studies could elucidate whether veterinary UC patients will respond favourably to anti-PD-1 immune checkpoint inhibitor therapy.

Evaluation of PD-1 and PD-L1 expression in canine urothelial carcinoma cell lines.

Urothelial carcinoma (UC) is the most common urinary tumor in dogs and despite combinational therapies, only modest improvements in survival have been achieved in recent years. Given the utility of monoclonal antibodies against PD-1 and PD-L1 in human UC, we evaluated the protein and mRNA expression in three established canine urothelial carcinoma cell lines. Flow cytometry and western blot analysis confirmed cell line expression of both molecules in varying degrees. Reverse transcription PCR (RT-PCR) documented mRNA expression in all three cell lines for both PD-1 and PD-L1. Fluorescence microscopy was consistent with strong PD-1 and PD-L1 expression in the canine cell lines and was in line with previous human literature. Importantly, the flow cytometry work described in this study revealed higher cell intrinsic PD-1 expression in these cell lines which may have implications for tumor behavior and potential treatment opportunities in the future. Further work is necessary to determine the expression patterns in canine UC and potential for benefit with immunotherapy directed against PD-1 and PD-L1.

MicroRNA profiling in canine multicentric lymphoma.

Lymphoma is the most common hematopoietic tumour in dogs and is remarkably similar to the human disease. Tumour biomarker discovery is providing new tools for diagnostics and predicting therapeutic response and clinical outcome. MicroRNAs are small non-coding RNAs that participate in post-transcriptional gene regulation and their aberrant expression can impact genes involved in cancer. The aim of this study was to characterize microRNA expression in lymph nodes and plasma from dogs with multicentric B or T cell lymphoma compared to healthy control dogs. We further compared expression between lymph nodes and corresponding plasma samples and assessed changes in expression at relapse compared to time of diagnosis. Lastly, we investigated microRNAs for association with clinical outcome in patients treated with CHOP chemotherapy. A customized PCR array was utilized to profile 38 canine target microRNAs. Quantification was performed using real time RT-qPCR and relative expression was determined by the delta-delta Ct method. In lymph nodes, there were 16 microRNAs with significantly altered expression for B cell lymphoma and 9 for T cell lymphoma. In plasma, there were 15 microRNAs altered for B cell lymphoma and 3 for T cell lymphoma. The majority of microRNAs did not have correlated expression between lymph node and plasma and only 8 microRNAs were significantly different between diagnosis and relapse. For B cell lymphoma, 8 microRNAs had differential expression in the non-remission group compared to dogs that completed CHOP in complete remission. Four of these microRNAs were also altered in patients that died prior to one-year. Kaplan-Meier survival curves for high versus low microRNA expression revealed that 10 microRNAs were correlated with progression-free survival and 3 with overall survival. This study highlights microRNAs of interest for canine multicentric lymphoma. Future goals include development of microRNA panels that may be useful as biomarkers with the intent to provide improved outcome prediction to veterinary cancer patients.

Retro-orbital and disseminated B-cell lymphoma in a yellow-collared macaw (Primolius auricollis).

A yellow-collared macaw was presented with unilateral left exophthalmia. The complete blood cell count and biochemistry revealed a heterophilic leukocytosis and elevation in liver parameters, respectively. A computed tomography scan showed a contrast-enhancing retrobulbar mass and hepatomegaly. Cytology of the liver was consistent with a round cell tumor, most likely lymphoma. The bird died after 2 months of palliative care. Postmortem examination confirmed a retro-orbital and disseminated B-cell lymphoma.

Lymphome B rétro-orbital et disséminé chez un ara à collier jaune(Primolius auricollis). Un ara à collier jaune a été présenté avec de l'exophtalmie unilatérale gauche. La formule sanguine complète et la biochimie ont révélé une leucocytose hétérophile et une élévation des paramètres hépatiques, respectivement. La tomodensitométrie à l'aide d'une injection de milieu de contraste a montré une masse rétrobulbaire et une hépatomégalie. La cytologie du foie était conforme à une tumeur à cellules rondes, le plus probablement un lymphome. L'oiseau est mort après 2 mois de soins palliatifs. L'examen postmortem a confirmé un lymphome B rétro-orbital et disséminé.(Traduit par Isabelle Vallières).

Assessment of platelet function in healthy cats in response to commonly prescribed antiplatelet drugs using three point-of-care platelet function tests.

Objectives The objective was to determine if decreased platelet function could be detected after treatment with aspirin and/or clopidogrel in healthy cats using three point-of-care platelet function tests that evaluate platelet function by different methods: Multiplate (by impedance), Platelet Function Analyzer 100 (by mechanical aperture closure) and Plateletworks (by platelet counting). Methods Thirty-six healthy cats were randomly assigned to receive one of three oral treatments over an 8 day period: (1) aspirin 5 mg q72h; (2) aspirin 20.25 mg q72h; or (3) clopidogrel 18.75 mg q24h. Cats treated with 5 and 20.25 mg aspirin also received clopidogrel on days 4-8. Platelet aggregation in response to adenosine diphosphate and collagen ± arachidonic acid was assessed on days 1 (baseline), 4 and 8. Aspirin and clopidogrel metabolites were measured by high-performance liquid chromatography. Platelet function in response to treatment was analyzed by ANCOVA, linear regression and Spearman correlation. Results The only solitary aspirin effect was detected using Plateletworks with collagen in cats treated with 20.25 mg. The only effect detected by Multiplate was using arachidonic acid in cats treated with both aspirin 20.25 mg and clopidogrel. All clopidogrel treatment effects were detected by Platelet Function Analyzer 100, Plateletworks (adenosine diphosphate) and Plateletworks (collagen). Drug metabolites were present in all cats, but concentrations were minimally correlated to platelet function test results. Conclusions and relevance Platelet Function Analyzer 100 and Plateletworks using adenosine diphosphate ± collagen agonists may be used to detect decreased platelet function in response to clopidogrel treatment. Either aspirin is not as effective an antiplatelet drug as clopidogrel, or the tests used were not optimal to measure aspirin effect. Cats with heart disease are commonly prescribed antiplatelet drugs to decrease the risk of aortic thromboembolism. Platelet Function Analyzer 100 and Plateletworks may be useful for confirming clopidogrel treatment in these cats.

Biologic variability and correlation of platelet function testing in healthy dogs.

BACKGROUND: Platelet function tests are influenced by biologic variability, including inter-individual (CVG ) and intra-individual (CVI ), as well as analytic (CVA ) variability. Variability in canine platelet function testing is unknown, but if excessive, would make it difficult to interpret serial results. Additionally, the correlation between platelet function tests is poor in people, but not well described in dogs. OBJECTIVES: The aims were to: (1) identify the effect of variation in preanalytic factors (venipuncture, elapsed time until analysis) on platelet function tests; (2) calculate analytic and biologic variability of adenosine diphosphate (ADP) and arachidonic acid (AA)-induced thromboelastograph platelet mapping (TEG-PM), ADP-, AA-, and collagen-induced whole blood platelet aggregometry (WBA), and collagen/ADP and collagen/epinephrine platelet function analysis (PFA-CADP, PFA-CEPI); and (3) determine the correlation between these variables. METHODS: In this prospective observational trial, platelet function was measured once every 7 days, for 4 consecutive weeks, in 9 healthy dogs. In addition, CBC, TEG-PM, WBA, and PFA were performed. RESULTS: Overall coefficients of variability ranged from 13.3% to 87.8% for the platelet function tests. Biologic variability was highest for AA-induced maximum amplitude generated during TEG-PM (MAAA; CVG = 95.3%, CVI = 60.8%). Use of population-based reference intervals (RI) was determined appropriate only for PFA-CADP (index of individuality = 10.7). There was poor correlation between most platelet function tests. CONCLUSIONS: Use of population-based RI appears inappropriate for most platelet function tests, and tests poorly correlate with one another. Future studies on biologic variability and correlation of platelet function tests should be performed in dogs with platelet dysfunction and those treated with antiplatelet therapy.

Assessment of platelet function in healthy sedated cats using three whole blood platelet function tests.

The objectives of this study were to establish feline references intervals for 3 commercial whole blood platelet function test analyzer systems: Multiplate analyzer (MP; Roche Diagnostics International Ltd., Rotkreuz, Switzerland), Platelet Function Analyzer-100 (PF: Siemens Canada, Mississauga, Ontario, Canada), and Plateletworks Combo-25 kit (PW; Helena Laboratories, Beaumont, TX). Venipuncture was performed on 55 healthy sedated cats, and platelet aggregation in response to adenosine diphosphate (ADP), collagen (COL), and arachidonic acid (AA; MP only) was assessed using citrated blood. For the MP analyzer, median (95% confidence intervals [CIs]) area under curve (Units) for ADP, COL, and AA agonists were 87 (11-176), 81 (32-129), and 91 (59-129), respectively. For the PF analyzer, median (95% CIs) closure time, using COL-ADP cartridges, was 69 (46-89) sec. For the PW assay, median (95% CIs) percent aggregations for ADP and COL agonists were 71 (18-92) and 49 (9-96), respectively, using impedance hematology analyzer platelet counts, and 94 (25-98) and 68 (14-119), respectively, using flow cytometry hematology analyzer platelet counts. There were low correlations between the PF analyzer (COL-ADP cartridge) and MP analyzer (COL agonist; ρ = 0.11), and between the PF analyzer (COL-ADP cartridge) and PW assay (COL agonist using impedance platelet counts; ρ = 0.14). The PW assay percent aggregations using impedance and flow cytometric platelet counts were correlated for both ADP (ρ = 0.64) and COL (ρ = 0.64) agonists. Platelet function testing using these tests are feasible in cats, but 95% CIs are wide, so single results may be difficult to interpret. Platelet counting by impedance or flow cytometry may be used for the PW assay but are not interchangeable.

Thromboelastography platelet mapping in healthy dogs using 1 analyzer versus 2 analyzers.

The objective of this study was to describe the results of thromboelastography platelet mapping (TEG-PM) carried out using 2 techniques in 20 healthy dogs. Maximum amplitudes (MA) generated by thrombin (MAthrombin), fibrin (MAfibrin), adenosine diphosphate (ADP) receptor activity (MAADP), and thromboxane A2 (TxA2) receptor activity (stimulated by arachidonic acid, MAAA) were recorded. Thromboelastography platelet mapping was carried out according to the manufacturer's guidelines (2-analyzer technique) and using a variation of this method employing only 1 analyzer (1-analyzer technique) on 2 separate blood samples obtained from each dog. Mean [± standard deviation (SD)] MA values for the 1-analyzer/2-analyzer techniques were: MAthrombin = 51.9 mm (± 7.1)/52.5 mm (± 8.0); MAfibrin = 20.7 mm (± 21.8)/23.0 mm (± 26.1); MAADP = 44.5 mm (± 15.6)/45.6 mm (± 17.0); and MAAA = 45.7 mm (± 11.6)/45.0 mm (± 15.4). Mean (± SD) percentage aggregation due to ADP receptor activity was 70.4% (± 32.8)/67.6% (± 33.7). Mean percentage aggregation due to TxA2 receptor activity was 77.3% (± 31.6)/78.1% (± 50.2). Results of TEG-PM were not significantly different for the 1-analyzer and 2-analyzer methods. High correlation was found between the 2 methods for MAfibrin [concordance correlation coefficient (r) = 0.930]; moderate correlation was found for MAthrombin (r = 0.70) and MAADP (r = 0.57); correlation between the 2 methods for MAAA was lower (r = 0.32). Thromboelastography platelet mapping (TEG-PM) should be further investigated to determine if it is a suitable method for measuring platelet dysfunction in dogs with thrombopathy.

Cette étude visait à décrire les résultats de la cartographie de la thromboélastographie des plaquettes (TEG-PM) effectuée à l'aide de deux techniques chez 20 chiens en santé. L'amplitude maximale (MA) générée par la thrombine (MAthrombine), la fibrine (MAfibrine), l'activité du récepteur de l'adénosine diphosphate (ADP) (MAADP), l'activité du récepteur de la thromboxane A2 (TxA2) (stimulée par l'acide arachidonique, MAAA) ont été mesurées. La TEG-PM a été effectuée selon les recommandations du manufacturier (technique à 2 analyseurs) ainsi qu'une variation de cette méthode en utilisant seulement un analyseur (technique à 1 analyseur) sur deux échantillons sanguins séparés obtenus de chaque chien. Les valeurs moyennes [± écart-type (SD)] de MA pour les techniques à 1 analyseur/2 analyseurs étaient: MAthrombine = 51,9 mm (± 7,1)/52,5 mm (± 8,0); MAfibrine = 20,7 mm (± 21,8)/23,0 mm (± 26,1); MAADP = 44,5 mm (± 15,6)/45,6 mm (± 17,0); et MAAA = 45,7 mm (± 11,6)/45,0 mm (± 15,4). La moyenne (± SD) du pourcentage d'agrégation due à l'activité du récepteur ADP était de 70,4 % (± 32,8)/67,6 % (± 33,7). La moyenne du pourcentage d'agrégation due à l'activité du récepteur TxA2 était de 77,3 % (± 31,6)/78,1 % (± 50,2). Il n'y avait pas de différence significative dans les résultats de TEG-PM entre les méthodes à 1 analyseur ou à 2 analyseurs. Une corrélation élevée a été trouvée entre les deux méthodes pour la MAfibrine [coefficient de concordance de corrélation (r) = 0,930]; une corrélation modérée a été trouvée pour MAthrombine (r = 0,70) et MAADP (r = 0,57); la corrélation entre les deux méthodes pour MAAA était plus faible (r = 0,32). Des études supplémentaires devraient être effectuées pour déterminer si la TEG-PM est une méthode qui convient pour mesurer le dysfonctionnement des plaquettes chez les chiens avec thrombopathie.(Traduit par Docteur Serge Messier).

Characterization of chicken thrombocyte responses to Toll-like receptor ligands.

Thrombocytes are the avian equivalent to mammalian platelets. In addition to their hemostatic effects, mammalian platelets rely in part on pattern recognition receptors, such as the Toll-like receptors (TLR), to detect the presence of pathogens and signal the release of certain cytokines. Ligands for TLRs include lipopolysaccharide (LPS), which is bound by TLR4, as well as unmethylated CpG DNA motifs, which are bound by TLR9 in mammals and TLR21 in chickens. Similar to mammalian platelets, avian thrombocytes have been shown to express TLR4 and secrete some pro-inflammatory cytokines in response to LPS treatment. However, the full extent of the contributions made by thrombocytes to host immunity has yet to be elucidated. Importantly, the mechanisms by which TLR stimulation may modulate thrombocyte effector functions have not been well characterized. As such, the objective of the present study was to gain further insight into the immunological role of thrombocytes by analyzing their responses to treatment with ligands for TLR4 and TLR21. To this end, we quantified the relative expression of several immune system genes at 1, 3, 8 and 18 hours post-treatment using real-time RT-PCR. Furthermore, production of nitric oxide and phagocytic activity of thrombocytes was measured after their activation with TLR ligands. We found that thrombocytes constitutively express transcripts for both pro- and anti-inflammatory cytokines, in addition to those associated with anti-viral responses and antigen presentation. Moreover, we found that both LPS and CpG oligodeoxynucleotides (ODN) induced robust pro-inflammatory responses in thrombocytes, as characterized by more than 100 fold increase in interleukin (IL)-1ß, IL-6 and IL-8 transcripts, while only LPS enhanced nitric oxide production and phagocytic capabilities. Future studies may be aimed at examining the responses of thrombocytes to other TLR ligands.

Evaluation of thrombelastographic platelet-mapping in healthy cats.

BACKGROUND: Thrombelastography (TEG) permits analysis of clot formation but it is not specific for platelet activity. TEG PlateletMapping (TEG-PM) is a modification of TEG that uses adenosine diphosphate (ADP) and arachidonic acid (AA) as platelet agonists to define the contribution of platelets to clot formation. OBJECTIVES: The objectives of this study were to determine values for TEG-PM in healthy cats and the interassay variation of TEG-PM. METHODS: TEG-PM analysis was performed on blood specimens collected from 12 healthy cats and was repeated using a second blood specimen collected 2 hours later. Maximum amplitudes generated by thrombin (MA(thrombin)), fibrin (MA(fibrin)), ADP-stimulated platelet activity (MA(ADP)), and AA-stimulated platelet activity (MA(AA)) were recorded. RESULTS: Mean ± SD for MA(thrombin) was 51.1 ± 8.5 mm, for MA(fibrin) was 32.3 ± 17.7 mm, for MA(ADP) was 32.3 ± 15.0 mm, and for MA(AA) was 24.5 ± 12.2 mm. Mean MA(ADP) and MA(fibrin) were not significantly different, whereas mean MA(AA) was significantly lower than mean MA(fibrin). Results from the first and second specimens were not significantly different. Correlation between the first and second specimens was moderate for MA(thrombin), MA(fibrin), and MA(ADP), but was poor for MA(AA). A high degree of variability (coefficient of variation 47.7-60.0%) was observed for MA(fibrin), MA(ADP), and MA(AA). CONCLUSIONS: As MA(ADP) and MA(AA) (AA) were the same as or lower than MA(fibrin), a valid baseline to determine platelet-stimulated clot formation could not be established. Considerable interassay variation and wide intervals for MA(fibrin), MA(ADP), and MA(AA) values in this study indicate that TEG-PM should be used cautiously in feline patients. Several preanalytical factors should be examined in further detail.

Effects of noncytopathic type 2 bovine viral diarrhea virus on the proliferation of bone marrow progenitor cells.

The purpose of this study was to investigate the effects of isolates of noncytopathic type 2 Bovine viral diarrhea virus (ncpBVDV-2) of high and low virulence on the proliferation of bone marrow progenitor cells. Holstein calves 6 to 7 mo old and BVDV-naïve were inoculated intranasally with a BVDV isolate of high virulence (HV24515), a BVDV isolate of low virulence (LV11Q), or uninfected cell culture medium. Serial bone marrow and peripheral blood samples were collected before and after inoculation. Bone marrow mononuclear cells (BMMCs) were isolated and cultured for 5 d, and the mean number of colony-forming unit-granulocyte-macrophage (CFU-GM) colonies was determined. Tritiated (3H)-thymidine uptake by BMMCs was determined to indicate overall proliferative capacity. Virus isolation was done on concurrent samples of BMMCs and peripheral blood. Virus was isolated from BMMCs and peripheral blood buffy-coat cells as early as day 2 or 3 after inoculation. Neutropenia developed in both groups inoculated with a BVDV isolate. However, in the calves given LV11Q, neutrophil counts rebounded earlier in response to increased proliferation of BMMCs, whereas the response was delayed in calves given HV24515. Thymidine uptake was significantly increased (P = 0.0047) in BMMCs after inoculation compared with before inoculation in the calves given LV11Q but not in those given HV24515 or in the control calves. The median number of CFU-GM colonies was significantly decreased (P = 0.0164) after inoculation compared with before inoculation in the calves given HV24515, whereas there was no significant difference in the calves given LV11Q or in the control calves. The data support the hypothesis that the prolonged neutropenia observed in calves given HV24515 results at least in part from decreased proliferative capacity of bone marrow progenitor cells.

Use of a commercial methylcellulose medium with and without recombinant bovine granulocyte colony-stimulating factor for culturing bovine bone marrow cells.

A commercial methylcellulose culture medium, with and without the addition of recombinant bovine granulocyte colony-stimulating factor (rbG-CSF), was utilized for culturing bovine bone marrow cells in a colony-forming unit assay. Bone marrow mononuclear cells were isolated and cultured in a commercial methylcellulose-based medium containing several recombinant human cytokines. Cultures were prepared with and without 100 ng/mL of rbG-CSF. The size and mean number of colonies per plate from culture days 3 to 9 were compared. We concluded that bovine bone marrow colony growth was supported by this culture medium. The addition of rbG-CSF yielded larger and more numerous colonies. There were significantly more colonies on day 3 (P < 0.001), day 4 (P < 0.001), and day 5 (P = 0.03) with rbG-CSF. Both culture media had the highest colony counts on day 5.

Effect on hematopoietic tissue of experimental infection of calves with noncytopathic type 2 bovine viral diarrhea virus.

To investigate the hematologic abnormalities observed with noncytopathic type 2 bovine viral diarrhea virus (ncpBVDV-2), calves 6 to 8 mo old were inoculated with an isolate of either high virulence (HV24515) or low virulence (LV11Q); control animals received the same volume of uninfected cell-culture supernatant. Peripheral blood neutrophil, lymphocyte, and platelet counts decreased in all the virus-inoculated calves but were significantly lower and remained decreased longer in the calves given HV24515. For each isolate, a decrease in the number of mature myeloid cells in the bone marrow coincided with the development of neutropenia, but the depletion persisted significantly longer (4 to 6 d) in the calves given HV24515. In the bone marrow of calves given LV11Q, the number of proliferating myeloid cells increased in proportion to the decrease in the number of mature myeloid cells. In the calves inoculated with HV24515, BVDV antigen was observed in bone marrow cells when the peripheral blood counts were lowest. Megakaryocytes were the predominant cell type exhibiting positive BVDV staining; myeloid cells rarely stained positively. Viral antigen was not observed in the bone marrow of calves given LV11Q. These experiments demonstrated that ncpBVDV-2 isolates of both high and low virulence caused decreased leukocyte and platelet counts, but only the high-virulence HV24515 isolate caused a delay in the production of myeloid proliferating cells. The delay may contribute to the ability of certain ncpBVDV-2 isolates to induce severe disease.