The influence of inflammation and hematocrit on clot strength in canine thromboelastographic hypercoagulability

Objective

To investigate parameters causing canine thromboelastographic hypercoagulability and to investigate whether thromboelastography (TEG) with Cytochalasin D (Cyt D) added is related to parameters of platelet activity.

Design

Prospective observational study on hemostatic and inflammatory parameters. Data were collected between November 2012 and July 2013.

Setting

University teaching hospital.

Animals

Twenty‐eight dogs suffering from diseases predisposing to thrombosis and 19 clinically healthy dogs. Diseased dogs were enrolled if they fulfilled inclusion criteria regarding age, size, informed client consent, and obtained a diagnosis of a disease that has been associated with thrombosis or hypercoagulability.

Interventions

None.

Measurements and Main Results

Parameters of coagulation and anticoagulation, fibrinolysis, and antifibrinolysis, platelet activity, inflammation, platelet count, and hematocrit were measured using CBC, TEG, platelet aggregation on multiplate, platelet activity on flow cytometry, and hemostatic and inflammatory markers on plasma and serum analyses. ANOVA and multilinear regression analyses indicated that especially hematocrit and the inflammatory parameters C‐reactive protein and interleukin‐8 showed best association with overall clot strength in diseased dogs with hypercoagulable TEG tracings. Ratios presumed to reflect platelet contribution to the TEG tracing obtained in TEG analyses with Cyt D were related especially with hematocrit and P‐selectin expression of platelets measured after γ‐Thrombin activation on flow cytometry.

Conclusion

Overall clot strength in TEG analyses of the hypercoagulable dogs included in the present study appears to be primarily associated with inflammation as well as hematocrit. Furthermore, the ratio between standard TEG analyses and TEG analyses with Cyt D may reflect some degree of platelet activity.     

Effect of rest temperature on rotational thromboelastometry in New Zealand White rabbits

With the increasing popularity of viscoelastic coagulation analyzers, such as rotational thromboelastometry [ROTEM] and thromboelastography, the need for standardized methodology for appropriate interpretation has become increasingly important. Viscoelastic analysis is heavily influenced by a multitude of pre-analytic factors, both in vivo and in vitro, leading to a large amount of variation between institutions. We investigated the effect of room temperature during a 30-min sample rest time on ROTEM, which analyzed both intrinsic and extrinsic pathways. We also evaluated the feasibility of using ROTEM to assess coagulation in non-anesthetized domestic rabbits. Rabbits were selected because they are a common companion animal that could benefit from the use of viscoelastic analysis for various disease processes that could lead to coagulopathies. Citrated whole blood was collected from 10 rabbits and allowed to rest upright for 30 min either at room temperature (~ 21°C) or in a tube warmer (37°C) before analysis. There was no significant difference in results between room temperature and warmed samples, which suggests that allowing samples to rest at room temperature is acceptable clinically. Additionally, blood collection and analysis were feasible in all rabbits.     

Effects of storage temperature and time on canine plasma ammonia concentrations

Stability of ammonia in canine plasma was determined, with regard to temperature and time of storage. Heparinized venous blood samples were collected from 8 healthy dogs, immediately placed in an ice water bath, and centrifuged at 5 C. Plasma was harvested from the blood samples, and the initial analysis of each sample for plasma ammonia was performed within 30 minutes after collection. Separate aliquots of the plasma from each dog were stored at 21 C, 4 C, -15 C, or -40 C. Ammonia concentrations of the aliquots stored at the various temperatures were determined at 24, 48, and 96 hours after collection. Statistical analysis of the data from each dog did not indicate a significant relationship between the initial concentration of plasma ammonia and subsequent determinations. Correlation or significance was not found among samples stored at similar temperatures and evaluated at similar times.     

Studies on immunoglobulins and trypsin inhibitor in colostrum and milk from sows and in serum of their piglets.

The concentrations of IgG, IgM, IgA and the specific sow colostrum trypsin inhibitor (SCTI) were measured by radial immunodiffusion in colostrum and milk samples from sows and in serum samples from their offspring during the suckling period. A clear time dependence was found for all the measured variates in both whey and serum. Statistically significant positive correlations were found between, on the one hand, concentrations of IgG and IgA, but not IgM, in sera from 39 suckling piglets 1 and 3 days old, and, on the other hand, concentrations of the same immunoglobulins and of the trypsin inhibitor in maternal colostrum (n = 7). Multiple regression analyses showed that at day 1 and day 3 the levels of both IgG and IgA in serum samples from the suckling piglets were positively influenced by both the SCTI and the IgG or IgA contents in maternal colostrum.     

Effects of sample handling on adrenocorticotropin concentration measured in canine plasma, using a commercially available radioimmunoassay kit.

A commercially available radioimmunoassay (RIA) kit for measurement of human adrenocorticotropin (hACTH) was validated for use in dogs. Assay sensitivity was 3 pg/ml. Intra-assay coefficient of variation (x 100; CV) for 3 canine plasma pools was 3.0 (mean +/- SD, 33 +/- 0.99 pg/ml), 4.2 (71 +/- 2.4 pg/ml) and 3.7 (145 +/- 3.7 pg/ml) %. Interassay CV for 2 plasma pools measured in 6 assays was 9.8 (37 +/- 3.6 pg/ml) and 4.4 (76 +/- 3.4 pg/ml) %, respectively. Dilutional parallelism was documented by assaying 2 pools of canine plasma at 3 dilutions and correcting the measured result for dilution. Corrected mean concentrations for the first pool were 33 (+/- 0.99), 36 (+/- 4.3), and 33 (+/- 6.8) pg/ml; corrected mean concentrations for the second pool were 145 (+/- 5.4), 141 (+/- 10.8) and 125 (+/- 3.4) pg/ml. Recovery of 1-39hACTH added to canine plasma (6.25, 12.5, 25.0, 50.0, and 100.0 pg/ml) was linear and quantitative (slope = 0.890, R2 = 0.961). To test whether anticoagulant or the protease inhibitor, aprotinin, influences ACTH concentration in canine plasma, ACTH was measured in canine blood collected in 4 tubes containing anticoagulant: heparin (H), heparin + 500 kallikrein inhibitor units (KIU) of aprotinin/ml (HA), EDTA (E), and EDTA + aprotinin (EA). Plasma ACTH concentration was the same when samples containing H and HA, or HA and E were compared, and was significantly (P less than 0.01) lower in samples containing EA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization, expression and function of c-Met in canine spontaneous cancers

Aberrant expression of the proto‐oncogene c‐Met has been noted in a variety of human cancers. To better define the potential role of Met dysregulation in canine cancer, the canine Met, hepatocyte growth factor (HGF) and HGF activator were cloned. Inappropriate expression of Met was present in canine tumour cell lines derived from a wide variety of cancers. Furthermore, both HGF and HGF activator were also expressed in several of these cell lines, providing evidence of a possible autocrine loop of Met activation. Stimulation of tumour cell lines with recombinant human HGF induced Met autophosphorylation, as well as activation of the downstream signalling elements Gab‐1, Akt and Erk1/2. Scattering of tumour cells and migration across a defect occurred in response to HGF stimulation. The Met inhibitor PHA665752 blocked both HGF‐induced phosphorylation of canine Met and HGF‐mediated cell cycling, scattering and migration. These studies provide evidence that Met dysregulation may play a role in the biology of canine cancer and lay the groundwork for future studies employing Met inhibitors.     

豆粕固态限定发酵和强化发酵的研究

本文采用短乳杆菌、酵母菌、枯草芽孢杆菌和地表芽孢杆菌进行豆粕固态限定发酵和强化发酵的研究,对发酵过程中小分子蛋白含量、菌量、pH及胰蛋白酶抑制剂活性等进行了测定.结果表明:纯菌条件下,接种短乳杆菌和地衣芽孢杆菌实验组限定发酵的小分子蛋白含量分别为31.0%和28.3%,高于接种酵母菌试验组的22.4%.且小分子蛋白积累高峰时间为48～72 h;接种地表芽孢杆菌实验组的胰蛋白酶抑制剂降解率高于接种短乳杆菌或酵母菌实验组.豆粕天然发酵试验组的小分子蛋白含量和胰蛋白酶抑制剂降解率分别为22.2%和44.1%.接种短乳杆菌、地衣芽孢杆菌试验组的小分子蛋白含量和胰蛋白酶抑制剂降解率分别为32.4%、99.5%和29.3%、99.7%.表明接种强化发酵有助于小分子蛋白含量的提高和胰蛋白酶抑制剂的降解.     

Double-blinded, placebo-controlled, cross-over pilot study on the efficacy of zileuton for canine atopic dermatitis

Nine dogs meeting the diagnostic criteria for canine atopic dermatitis were enrolled in a double-blind, placebo-controlled, cross-over clinical trial. In this pilot study, zileuton (a 5-lipoxygenase inhibitor) given orally at 2 mg kg(-1) three times daily for 4 weeks significantly decreased erythema in dogs with atopic dermatitis but had no effect on pruritus. Zileuton was well tolerated and no adverse clinical signs were noted. However, one dog developed mild alanine aminotransaminase elevation, which resolved within 1 week of discontinuation of therapy. Monitoring of alanine aminotransaminase may be necessary in dogs receiving zileuton. Further studies with larger number of dogs are needed to evaluate the efficacy of zileuton as treatment for canine atopic dermatitis.     

一年陈玉米和烘干玉米对种鸡蛋品质、血清抗氧化能力、繁殖性能和营养物质消化吸收效率的影响

为探究饲喂一年陈玉米和烘干玉米对种鸡蛋品质、血清抗氧化能力、繁殖性能和营养物质消化吸收效率的影响,试验选取224只55周龄海兰褐种鸡,随机分为一年陈玉米组和烘干玉米组,每组7个重复,每个重复16只鸡.试验期为56d.结果显示:与烘干玉米组相比,一年陈玉米组显著提高了种蛋蛋形指数、蛋白高度和蛋壳厚度(P＜0.05),显著...     

Effects of physiologic agonists on canine whole blood flow cytometry assays of leukocyte-platelet aggregation and platelet activation

Platelets play a role in both the innate and adaptive immune systems. Methods for detecting activated platelets and leukocyte-platelet aggregates (LPAs) are useful for basic and applied research concerning the role of platelets in inflammation and immune disorders. The aim of the study was to develop flow cytometric assays for detection of platelets binding to monocytes and neutrophils and for activated platelets in canine whole blood and to investigate the effect of physiologic agonists. Citrate anticoagulated whole blood was incubated with monoclonal antibodies against CD14 and CD61 for detection of LPAs, and the effect of various agonists was investigated. For detection of activated platelets, whole blood was incubated with monoclonal antibodies against CD62P and against a receptor-induced binding site on fibrinogen (CAP1) with CD61 as a platelet identifier. Isotype controls were prepared in parallel. The individual physiologic agonists ADP, collagen and epinephrine increased LPAs, CD62P and CAP1 binding only modestly. However, combinations of agonists gave more substantial increases. A dose-response relationship was seen using alpha- and gamma-thrombin, and ADP as agonists. In conclusion, we have developed flow cytometry assays to measure LPAs and platelet activation in canine whole blood, and have explored the effect of various physiologic agonists at different concentrations.     

Effects of storage time and freezing temperature on clinical chemical parameters from canine serum and heparinized plasma

To assess changes in 24 blood constituents in frozen serum and heparinized plasma, blood samples were drawn from 10 clinically normal German Shepherd army dogs. The storage characteristics of nine enzymes (ALP, ALT, amylase, AST, CK, GGT, GLDH, LDH, lipase), and 15 metabolites and minerals (albumin, bile acids, bilirubin, calcium, cholesterol, creatinine, fructosamine, glucose, magnesium, phosphate, potassium, protein, sodium, triglycerides, urea) were studied. Parallel samples of serum and heparinized plasma were stored for 90 and 240 days at two different storage temperatures, -200 degrees C and -700 degrees C. Sixteen of the 24 analytes (ALP, ALT, amylase, AST, CK, GGT, GLDH, LDH, bile acids, calcium, cholesterol, creatinine, fructosamine, magnesium, phosphate, urea) showed statistically significant (p < 0.05) changes during the storage period related to storage time, storage temperature, and sample type. Seven of the analytes (amylase, GGT, GLDH, LDH, bile acids, fructosamine, magnesium) showed changes of possible clinical importance with mean differences from baseline larger than 20% for the enzymes and 10% for the metabolites and minerals during the storage periods.     

Effects of urinary tract inflammation and sample blood contamination on urine albumin and total protein concentrations in canine urine samples

BACKGROUND: Urinary tract inflammation and hemorrhage are believed to be common causes of proteinuria in dogs based on results of studies that measured total urine protein concentration. A method to quantify urine albumin (UAlb) concentration in dogs recently has become available; however, the effect of inflammation on albuminuria is unknown. OBJECTIVES: The goals of this study were to determine the effects of urinary tract inflammation, as indicated by pyuria and sample blood contamination, on UAlb concentration and on urine protein:creatinine (UPC) ratio in dogs. METHODS: Urine samples were obtained from dogs with pyuria that were presented to a veterinary teaching hospital or were part of a laboratory colony. To mimic the effects of hematuria, canine whole blood was added to a microscopically normal canine urine sample that had baseline albumin and total protein concentrations below the limits of detection. UAlb concentration was measured using a canine albumin-specific competitive ELISA. UPC ratio was determined using routine methods. RESULTS: Of 70 samples with pyuria, 67% had negligible UAlb concentrations and 81% had normal UPC ratios. UAlb concentration but not UPC ratio was significantly higher (P < 0.05) in samples with concurrent hematuria or bacteriuria. When whole blood was added to normal urine, UAlb concentration did not exceed 1 mg/dL until the sample became visibly pink; the UPC did not exceed 0.4 at any dilution. CONCLUSIONS: Many dogs with pyuria do not have albuminuria or proteinuria; however, albuminuria may be more likely in dogs with pyuria and concurrent hematuria or bacteriuria. Hematuria may not cause an increase in UAlb concentration until it becomes macroscopic and even then may not increase the UPC ratio.     

乳酸菌对低温下玉米秸秆黄贮发酵品质及有氧稳定性的影响

为研究低温下乳酸菌对玉米秸秆黄贮发酵品质及有氧稳定性的影响,试验采用小规模发酵法,以收获籽粒后的玉米秸秆作为原料,分别添加乳酸菌LAB_1(Lactobacillus plantarum)、LAB_2(Lactococcus lactis)、LAB_3(Lactobacillus paracasei)、LAB_1+LAB_2、LAB_1+LAB_3、LAB_2+LAB_3,在低温(4℃)条件下发酵30 d后开封,测定发酵参数、微生物数量及有氧稳定性。结果表明:与对照组相比,添加乳酸菌可以降低pH和NH3-N含量(P <0.05),提高乳酸含量(P <0.05),抑制大肠杆菌、丁酸菌、霉菌等有害微生物的生长;在乳酸菌添加的各组间,pH从低到高依次为:LAB_3组 0.05),LAB_3组的乳酸含量较LAB_1、LAB_2、LAB_1+LAB_2、LAB_1+LAB_3、LAB_2+LAB_3组分别提高了11.85%、7.09%、3.42%、13.53%、4.86%,但无显著差异(P> 0.05),LAB_3组的氨态氮含量显著低于LAB_1组(P <0.05)。大肠杆菌数量从低到高依次是:LAB_3组相似文献