An enzyme immunoassay to measure canine circulating fibronectin

A competitive enzyme immunoassay has been used to detect and quantitate fibronectin in canine plasma. In this test, purified fibronectin, bound to microtiter plates, competes with plasma fibronectin for the conjugated antibody, rabbit-anticanine, fibronectin-horseradish peroxidase. The assay could detect fibronectin in purified standards from 58 ng/ml to 580 microgram/ml. The range of 1-100 microgram/ml was linear for plasma samples diluted 1:10, allowing samples with fibronectin concentrations from 10-1000 microgram/ml to be easily measured by this method. The mean normal fibronectin concentration of 132 dogs, by this method, was determined to be 320 +/- 74 microgram/ml.     

Deposition of fibronectin in articular cartilage of canine osteoarthritic joints

Deposition of fibronectin was examined in articular cartilage of healthy and osteoarthritic joints of Labrador Retriever dogs. Abnormal cartilage from osteoarthritic joints contained up to 20 times more fibronectin than did disease-free cartilage. Compared with normal cartilage, the rate of fibronectin synthesis in osteoarthritic cartilage was increased and a greater proportion of the newly made fibronectin accumulated in the lesion cartilage matrix. The rate of release of endogenous prelabeled [3H]fibronectin from disease-free explants was similar to the rate of release from degenerated cartilage explants into incubation media. Concentrations of fibronectin were greater in synovial fluid from osteoarthritic joints than in synovial fluid from healthy joints, but both types of fluid had less fibronectin than in blood plasma.     

Plasma fibronectin concentration associated with various types of canine neoplasia

Fibronectin, a large glycoprotein found in soluble form in plasma and in insoluble form in connective tissue matrices, has been implicated in cell-to-cell and cell-to-substratum interactions, inflammation and tissue repair, phagocytosis, hemostasis, and oncogenic cell transformation. Because fibronectin concentration is diminished or lacking on cell surfaces of many transformed cell lines and because decreased concentration of plasma fibronectin is associated with suboptimal mononuclear phagocyte system function and host defense, plasma fibronectin concentration was evaluated in 119 dogs with various forms of neoplasia. Included were 43 dogs with neoplasia of the skin and soft tissue, 18 with gastrointestinal tract neoplasia, 29 with mammary gland neoplasia, and 29 with various other types of neoplasia. Of the dogs studied, 44 (37%) had evidence of metastatic disease. This group had fibronectin concentration that differed significantly (P less than 0.01) from the plasma fibronectin concentration reference interval. Within this group, 9 dogs (20% of this group) had plasma fibronectin values within the reference interval, 33 (75%) had significantly (P less than 0.01) lower values than the reference interval, and 2 (5%) had significantly (P less than 0.01) higher values than the reference interval. These data suggested that fibronectin concentration determination, when results are abnormal, may be of diagnostic and prognostic interest.     

Detection of catecholamines and metanephrines by radio-immunoassay in canine plasma

This study investigated the applicability of two human radio-immunoassays (RIA) to detect epinephrine (EPI), norepinephrine (NE), and their O-methylated metabolites metanephrine (MN) and normetanephrine (NMN) in canine plasma. The analysis yielded a positive correlation between metabolites and their respective parent compounds: EPI and MN (r = 0.63), NE and NMN (r = 0.47), as well as between parent compounds, EPI and NE (r = 0.48), and between metabolites MN and NMN (r = 0.71). Moreover, EPI (r = 0.99) and NE (r = 0.77) concentrations determined by RIA did correlate positively with high pressure liquid chromatography (HPLC). However, there was limited agreement between both methods. It was concluded that complete validation tests for accuracy, precision and agreement are needed before this RIA can be applied to quantify catecholamines, metanephrine, and normetanephrine in canine plasma. The assay may prove to be a potential alternative to HPLC or tandem mass spectrometry in the work-up of pheochromocytoma and the detection of overall sympathetic activity in dogs.     

Quantitative analysis of canine plasma lipoproteins

A combined ultracentrifugationl/precipitation method for the measurement of lipoprotein cholesterol concentrations was developed and validated for use with canine plasma. Very low density lipoproteins (VLDL) were isolated by flotation ultracentrifugation and low density lipoproteins (LDL) separated from high density lipoproteins (HDL) by precipitation with heparin-manganese chloride. Effective separation of these classes was confirmed by agarose gel electrophoresis of native lipoproteins and by sodium dodecyl sulphate polyacrylamide gel electrophoresis of their apolipoprotein distributions. There was trace contamination of the LDL precipitate with HDL, but this represented less than 4 and 9 per cent of the total plasma HDL in normo- and hypercholesterolaemic dogs, respectively. The intra-assay and interassay coefficients of variation for LDL- and HDL-cholesterol concentrations were between 3·3 and 6·9 per cent, and 7·2 and 9·0 per cent, respectively, for plasma cholesterol concentrations between 2·67 and 8·14 mmoll/litre. The intra-assay coefficient of variation for VLDL-cholesterol was 53·8 and 18·4 per cent at plasma cholesterol concentrations of 2·67 and 8·14 mmol/litre, respectively. The interassay coefficient of variation for VLDL was 22·5 per cent. Storage of plasma at -20°C for between two and eight weeks did not affect VLDL-cholesterol concentrations, but led to an increase in LDL-cholesterol and a decrease in HDL-cholesterol concentrations of approximately 10 per cent. The method described is appropriate for the measurement of lipoprotein concentrations in plasma from normo- and hypercholesterolaemic dogs, but samples should not be subjected to prolonged storage before analysis.     

Cellulose acetate electrophoresis of canine plasma after fibrinogen precipitation by ethanol

Background: In routine canine medicine, anticoagulated blood is often the only sample sent to laboratories for diagnostic purposes. This hampers the interpretation of protein electrophoretic tracings because plasma contains fibrinogen, which migrates in the β–γ region. In human medicine, fibrinogen can be precipitated from plasma using ethanol. Objectives: The purpose of this study was to assess ethanol precipitation as a method for removing fibrinogen from canine plasma so as to facilitate the interpretation of electorphoresis results. Methods: Blood samples collected from 40 dogs were divided into plain tubes and tubes containing EDTA (n=20) or lithium–heparin (n=20). An aliquot of plasma from each sample was incubated with ethanol at a final concentration of 100 mL/L. Cellulose acetate electrophoresis was then performed on serum, plasma, and plasma treated with ethanol. To verify the efficiency of ethanol treatment, fibrinogen was added to 5 canine serum samples at final concentrations of 2.5, 5.0, and 10.0 g/L, and electrophoresis was performed before and after ethanol treatment. Results: Visual analysis of electrophoretograms from ethanol‐treated samples confirmed the disappearance of the fibrinogen peak from the β₂‐globulin region. Treatment with ethanol caused a significant decrease in the percentage of β₂‐globulins and a significant increase in the percentage of α₂‐globulins. Absolute values of most electrophoretic fractions were significantly decreased in ethanol‐treated plasma compared with serum. Conclusions: Ethanol treatment successfully removed fibrinogen from canine plasma and normalized electrophoretic profiles, but probably also precipitated proteins other than fibrinogen. Ethanol treatment is recommended to facilitate visual identification of abnormal monoclonal peaks, but not for determining absolute protein concentrations in electrophoretic fractions.     

Cytoplasmic blood plasma inclusions of canine hepatocytes demonstration by immunoperoxidase-labeling method

Cytoplasmic inclusions in canine hepatocytes were scattered abundantly throughout hepatic lobules. They were mainly round, measured 3 microns to 20 microns in diameter, stained eosinophilic with hematoxylin and eosin stain, and were positive for diastase-resistant periodic acid-Schiff and phosphotungstic acid hematoxylin stains. Immunoperoxidase staining revealed that most of the inclusions were positive for such blood plasma components as albumin, immunoglobulins G, M, A, and transferrin. Ultrastructurally, the inclusions were granular, floccular, fibrillar, or homogeneous with a low or high electron density. Fibrin fibers and erythrocytes were occasionally recognized in the inclusions. These inclusions were considered to have derived from engulfment of blood elements into hepatocytes, probably as a result of increase of the intrasinusoidal blood pressure.     

Effects of three antiarthritic drugs on fibronectin and keratan sulfate synthesis by cultured canine articular cartilage chondrocytes.

Because articular chondrocytes are a target for drugs that can influence the integrity of cartilage, we investigated the effects of 3 antiarthritic drugs, glycosaminoglycan polysulfate, diclofenac-Na, and S-adenosylmethionine sulfate p-toluenesulfonate on total protein, fibronectin, and DNA synthesis, as well as on extradomain-A fibronectin and keratan sulfate content. Glycosaminoglycan polysulfate stimulated dose-dependent incorporation of [35S]methionine into protein and fibronectin, whereas incorporation of [3H]thymidine into DNA was unaffected. Total fibronectin, extradomain-A fibronectin, and keratan sulfate content were high in chondrocyte cultures treated with glycosaminoglycan polysulfate. In contrast, fibronectin and DNA synthesis, as well as extradomain-A fibronectin and keratan sulfate content were unaffected by diclofenac-Na. S-Adenosyl-methionine decreased dose-dependently the synthesis of fibronectin, as well as the content of fibronectin and keratan sulfate. At the highest concentration of S-adenosyl-methionine tested, findings suggest that cell viability was impaired as assessed by the release of lactate dehydrogenase into the media.     

Diagnostic efficacy of plasma ACTH-measurement by a chemiluminometric assay in canine hyperadrenocorticism

This retrospective study was performed to investigate the diagnostic efficacy of the chemiluminometric ACTH-measurement to differentiate between pituitary and adrenal dependent hyperadrenocorticism (HAC) in dogs. 49 dogs with pituitary HAC, 10 dogs with adrenal HAC and 1 dog with a combination of both pathologies were included. Dogs with HAC like symptoms, where HAC had been ruled out, served as controls (n = 18). All dogs with adrenal HAC, as well as 9 dogs with pituitary HAC had an ACTH concentration below the detection limit of 2.2 pmol/l (10 pg/dl) plasma. Using 2.2 pmol/l as a cut-off the sensitivity and specificity to diagnose pituitary HAC was 0.82 (95 % CI 0.686 - 0.914) and 1 (95 % CI 0.692 - 1), respectively. With the help of the chemiluminometric assay, a correct classification was possible in 85 % of patients with HAC. As an ACTH-concentration below the detection limit was found in dogs with adrenal as well as pituitary HAC, additional discriminatory tests are necessary in these cases.     

Evaluation of microwave-thawed canine plasma for transfusion

Units of fresh-frozen canine plasma were thawed rapidly in a microwave oven, using a mean of 15.4 thawing periods of 10 seconds each. The activated partial thromboplastin times, the one-stage prothrombin times, concentrations of fibrinogen, factor-VIII coagulant activity, and von Willebrand factor antigen of microwave-thawed units were not significantly different from those measured in small aliquots of the same units thawed in a warm water bath (n = 5). Five dogs were given a unit of their own microwave-thawed plasma. During the 24 hours after infusion, no significant changes were measured in temperature, heart rate, or respiration rate. In addition, significant changes in PCV, total protein concentrations, estimates of platelet numbers, total RBC counts, total WBC counts, and differential WBC counts were not measured in blood specimens obtained before infusion and 24 hours after the infusion of microwave-thawed plasma.     

Post-prandial changes in canine plasma creatinine

Post-prandial changes of plasma creatinine and urea concentrations were found in healthy Beagle dogs fed a pelleted diet. Changes of plasma creatinine determined using an alkaline picrate method were confirmed by a more specific enzymatic method. The study also showed that alkaline picrate methods over-estimate plasma creatinine in dog plasma.     

Stability of stored canine plasma for hemostasis testing

BACKGROUND: A review of the literature revealed limited information about the stability of samples for coagulation testing in dogs. OBJECTIVE: The aim of this study was to evaluate the stability of individual coagulation factors, clotting times, and other parameters of hemostasis in stored canine plasma. METHODS: Citrated plasma samples were obtained from 21 dogs. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen concentration, and factor I, II, V, VII, VIII, IX, X, XI, and XII activities were measured on an automated coagulation analyzer with commercially available reagents. Antithrombin (AT) activity and D-dimer concentration were measured on an automated chemistry analyzer using validated kits. Samples were analyzed within 1 hour after collection (initial analysis) and once daily for 2 or 4 consecutive days following storage at room temperature (RT) or 4 degrees C, respectively. RESULTS: Storage time at either temperature did not have any effect on PT, factor II, V, VII, X, or XII activities, D-dimer concentration, or AT activity. In contrast, aPTT was significantly prolonged after 72 and 96 hours at 4 degrees C; fibrinogen concentration was decreased after 48 hours at RT; the activities of factors VIII and IX were decreased after 48, 72, and 96 hours at 4 degrees C; and factor XI activity was decreased after 72 hours at 4 degrees C. CONCLUSIONS: Results suggest that storage of canine plasma for 2 days at RT does not have a significant effect on hemostasis test results with the exception of a slight decrease in fibrinogen concentration. In contrast, aPTT and factors VIII, IX, and XI were unstable in refrigerated plasma after 48 or 72 hours of storage.     

犬新鲜冰冻血浆的制备及应用

从供血犬的选择、血液采集、血液成分分离、生物安全监测、运输等几个方面,初步建立了犬新鲜冰冻血浆制备的操作流程。结果显示:按此流程操作,供血犬采血后未出现不良反应;制备的犬新鲜冰冻血浆在临床上应用效果良好,除出现少数发热和过敏情况外,未发现严重输血反应及生物安全方面的医疗事故。