Effects of ovariohysterectomy on canine blood neutrophil respiratory burst: a chemiluminescence study

OBJECTIVE: To examine blood neutrophil counts and luminol-enhanced chemiluminescence (CL) responses in dogs undergoing ovariohysterectomy (OH), premedicated with 2 different drugs. STUDY DESIGN: Randomized clinical study. ANIMALS: Forty-two healthy client-owned bitches. METHODS: Dogs had OH under isoflurane anesthesia with either acepromazine or medetomidine, both in combination with butorphanol, administered as preanesthetic medication. Blood samples were collected when the dog was admitted, at the end of surgery, and the next day (approximately 20 hours after surgery). Blood neutrophils were counted automatically, and neutrophil oxidative activity was assessed by measuring blood CL responses (induced by opsonized zymosan and enhanced by luminol) at 37 degrees C for 40 minutes. RESULTS: Number of circulating neutrophils was significantly increased the day after surgery reflected by enhanced blood CL responses. Neutrophil CL, however, was not significantly altered. No significant differences were detected for perioperative Polymorphonuclear neutrophil (PMN) characteristics between the 2 preanesthetic regimens. CONCLUSIONS: In conclusion, despite clearly increasing the number of circulating neutrophils, OH did not significantly affect neutrophil respiratory burst, as measured by whole-blood CL responses. CLINICAL RELEVANCE: Surgical operation of moderate intensity (e.g., OH) did not significantly alter one of the important immune functions, neutrophil oxidative activity. Further studies are warranted to confirm the significance of this finding, and to assess the value of following this variable in different animal patient populations.     

Combined phagocytosis-nitroblue tetrazolium reduction test in bovine neutrophils

An assay was developed for the simultaneous evaluation of phagocytosis and oxidative metabolism of bovine blood neutrophils. Phagocytosis was evaluated by using opsonized zymosan, and oxidative metabolism was evaluated by nitroblue tetrazolium (NBT) reduction. Normal bovine neutrophils exhibited moderate variation in ability to phagocytize zymosan, but little variation in ability to reduce NBT. The subcellular location of NBT reduction to formazan was determined by electron microscopy. Electron dense formazan precipitate was observed along the inner membrane of phagosomes enclosing zymosan particles and radiating from the membrane toward the center of the phagosome.     

Comparison of radioimmunoassay and chemiluminescent assay methods to estimate canine blood cortisol concentrations

Background Non‐radioactive assay methods are widely used in commercial laboratories to measure canine blood cortisol concentrations, despite a paucity of published validity data of these tests compared with the traditional ‘gold standard’ radioimmunoassay. Objectives To compare a commercial chemiluminescence assay with radioimmunoassay for blood cortisol measurement, determine the effect of storage on the radioimmunoassay, and determine the impact of any differences on clinical decisions. Methods The study included 54 client owned dogs undergoing adrenal function testing. Fresh plasma or serum samples (n = 170) were assayed for cortisol using radioimmunoassay (RIA1). Samples (n = 196) were also frozen and stored in batches, and assayed by chemiluminescence and radioimmunoassay (RIA2). Results Overall, there was a strong correlation (r² = 0.967, P < 0.001) between RIA2 and chemiluminescence concentrations without significant difference between means. Strong correlations were present for RIA2 and chemiluminescence at concentration subgroups of > 400 nmol/L (r² = 0.869, P < 0.001), < 100 nmol/L (r² = 0.790, P < 0.001), and < 40 nmol/L (r² = 0.738, P < 0.001). Significant differences between means were present for RIA2 and chemiluminescence concentrations in the < 100 nmol/L, and < 40 nmol/L (P < 0.001) groups. Despite a significant difference in RIA1 and RIA2 results overall, there was no significant difference between RIA1 and RIA2 for any of the concentration groups. In seven cases, discrepant RIA2 and chemiluminescence results may have altered clinical decisions. Conclusions Although RIA and chemiluminescence cortisol concentrations appear highly correlated, a significant difference may exist for concentrations less than 100 nmol/L in stored canine sera. Results of chemiluminescence cortisol assays should be interpreted with caution unless the specific assay method in the laboratory has been adequately validated in dogs.     

Evaluation of neutrophil oxidative metabolism in canine monocytic ehrlichiosis

BACKGROUND: Canine monocytic ehrlichiosis (CME) is a tick-borne disease caused by Ehrlichia canis, a rickettsia that infects the monocytes of dogs. This infection can result in a chronic and life-threatening disease. Thrombocytopenia, mild anemia, and leukopenia are the most common hematologic findings in CME. OBJECTIVE: To investigate the role of peripheral blood neutrophils in CME, an evaluation was conducted of their functional state during the acute phase of the disease in dogs experimentally infected by E canis. METHODS: Seven dogs were inoculated with E canis, and 3 remained as uninfected controls. All dogs had physical exams and hematologic tests (CBC and nitroblue tetrazolium [NBT] reduction) during a 6-week period. RESULTS: There was no difference (P > .05) in spontaneous NBT reduction results between the 2 groups of dogs throughout the 6-week period of observation. Nevertheless, when stimulated, the neutrophils showed higher activity in the infected group (P = .01) on weeks 4 and 5 after infection. CONCLUSION: Infection by E canis has no influence on neutrophil oxidative metabolism even though during the remission period of the acute phase of the disease, the neutrophils seem to be more reactive under stimulation.     

Factors affecting the adherence of Malassezia pachydermatis to canine corneocytes in vitro

Abstract Suspensions of Malassezia pachydermatis adhered to canine corneocytes attached io adhesive tape in a dose (P < 0.001) and time-dependent (P < 0.01) manner; adherence was maximal after 2 h. M. pachydermatis cells were approximately 10 times more adherent than Saccharomyces cerevisiae (P < 0.001) cells after 2 h incubation. The adherence of formalin-treated and frozen-thawed M. pachydermatis cells was comparable with untreated controls. Stationary-phase cells adhered better (P < 0.05) than exponential-phase cells. Pretreatment of the yeasts, or corneocytes, with 0.1% trypsin for 30 min reduced (P < 0.01) the adherence of four, and two, out of five strains, respectively, whereas incubation with 300 mM solutions of D(+) mannose, sucrose and N-acetyl D-glucosamine had no consistent effect. These results suggest that trypsin-sensitive proteins or glycoproteins on the yeast cell wall, and on the corneocyte surface, play an important role in the adherence of M. pachydermatis to canine corneocytes in vitro, whereas a role for carbohydrate receptors was not demonstrated. Résumé— Des suspensions de Malassezia pachydermatis adhérant à des cornéocytes des chiens sont attachées à des rubans adhésifs de façon significative en function du nombre (P < 0,001) et de la durée (P < 0,01). L'adhérence est maximale après 2 heures. Les levures du genre Malassezia pachydermatis sont approximativement dix fois plus adhérentes que les levures du genre Saccharomyces cerevisiae (P < 0,001) après une incubation de 2 heures. L'adhérence des Malassezia pachydermatis traitées par le formol et congelées - décongelées, est comparable à celle des témoins. Les levures qui ne sont pas en phase de croissance adhérent mieux (P < 0,05) que celles qui le sont. Le traitement préalable des levures ou des cornéocytes avec une solution de trypsine pendant 30 minutes réduit (P < 0,01) l'adhérence tandis que l'incubation avec des solutions à 300 mM de D + mannose, sucrose et de N acétyl D glucosamine n'a pas d'effets. Ces résultats suggèrent que des protéines sensibles à la trypsine ou des glycoprotéines sur la paroi des levures et à la surface des cornéocytes jouent un rôle important in vitro dans l'adhérence des Malassezia pachydermatis aux cornéocytes du chien, alors que le rôle des récepteurs glucidiques n'a pas été démontré. [Bond, R., Lloyd, D. H. Factors affecting the adherence of Malassezia pachydermatis to canine cornéocytes in vitro (Facteurs influençant l'adhérence de Malassezia pachydermatis aux cornéocytes du chien in vitro). Veterinary Dermatology 1996; 7 : 49–56.] Resumen Las suspensiones de Malassezia pachydermatis se adherian a cinta adhesiva de forma dependiente de la dosis (P < 0.001) y del tiempo (P < 0.01); su adherencia fue máxima a las 2 h. Las células de M. pachydermatis fueron aproximadamente 10 veces más adherentes que las de Saccharomyces cerevisiae (P < 0.001) a las 2 h de incubación. La adherencia de células de M. pachydermatis tratadas con formalina y congeladas-descongeladas fue comparable con los controles no tratados. Las células en estadio estacionario se adherian mejor (P < 0.05) que las de fase exponencial. El tratamiento previo de las levaduras o los corneocitos con 0.1% de tripsina durante 30 min redujo (P < 0.01) la adherencia de cuatro, y dos, de cinco cepas, respectivamente, mientras que su incubación con soluciones 300 mM de D(+) manosa, sucrosa y N-acetil D-glucosamina no tuvieron un efecto constante. Estos resultados sugieren que proteinas o glieoproteinas sensibles a la tripsina en la pared de la levadura, y en la superficie del corneocito, juegan un papel importante en la adherencia de M. pachydermatis a los corneocitos caninos in vitro, mientras que no se pudo demostrar un papel por parte de los receptores de carbohidratos. [Bond, R., Lloyd, D. H. Factors affecting the adherence of Malassezia pachydermatis to canine corneocytes in vitro (Facto res que afectan la adherencia de Malassezia pachydermatis a los corneocitos caninos in vitro). Veterinary Dermatology 1996; 7 : 49–56.] Zusammenfassung— Suspensionen von Malassezia pachydermatis zeigten eine Adhärenz an kanine Korneozyten, die an einem Klebeband befestigt waren, in dosisabhängiger (P < 0,001) und zeitabhängiger Weise (P < 0,01). Die Adhärenz erreichte ein maximum nach 2 Stunden. M. pachydermatis-Zellen waren ungefähr 10 mal stärker adhärent als Saccharomyces cervisiae-Zellen nach Zstündiger Inkubation (P < 0,001). Die Adhärenz von formalinbehandelten und gefrorenen/aufgetauten M. pachydermatis-Zellen war vergleichbar mit unbehandelten Kontrollzellen. Zellen der stationären Phase waren besser adhärent (P < 0,05) als Zellen der exponentiellen Phase. Eine Vorbehandlung der Hefen oder Korneozyten mit 0,1% igem Trypsin über 30 Minuten reduzierte die Adhärenz (P < 0,01) von 4 bzw. 2 aus 5 Linien, während eine Inkubation mit 300 mm Lösungen von D(+)Mannose, Sucrose und N-Acetyl-D-Glukosaminen keinen entsprechenden Effekt hatte. Diese Ergebnisse legen nahe, daß Trypsin-sensible Proteine oder Glykoproteine an der Zellwand der Hefe und auf der Korneozytenoberfläche eine wichtige Rolle für die Adhärenz von M. pachydermatis an kanine Korneozyten in-vitro spielen, während eine Bedeutung für Kohlenhydrat-Rezeptoren nicht demonstriert werden konnte. [Bond, R., Lloyd, D. H. Factors affecting the adherence of Malassezia pachydermatis to canine corneocytes in vitro (Einflußfaktoren auf die Adhärenz von Malassezia pachydermatis an kanine Korneozyten in vitro). Veterinary Dermatology 1996; 7 : 49–56.]     

In vitro heparinization of canine whole blood with low molecular weight heparin (dalteparin) significantly and dose‐dependently prolongs heparinase‐modified tissue factor‐activated thromboelastography parameters and prothrombinase‐induced clotting time

Background: Low‐molecular‐weight heparin (LMWH) is being used increasingly in veterinary medicine for both treatment and prophylaxis of thromboembolic disease, but no predictable patient‐side method exists to monitor its effect. Objectives: The aim of this study was to evaluate thromboelastography (TEG) and prothombinase‐induced clotting time (PiCT) assays for detecting hemostatic alterations following in vitro heparinization of canine whole blood with dalteparin (Fragmin). Methods: Citrated whole‐blood samples were collected from 7 clinically healthy dogs. Dalteparin was added at concentrations of 0, 0.156, 0.625, 1.25, and 2.5 U/mL of whole blood. TEG was performed using heparinase cups with tissue factor (TF, 1:50,000) and kaolin as activators. Reaction time (R), clotting time (K), angle (α), and maximum amplitude (MA) were recorded. PiCT and anti‐FXa activity were measured in plasma. Results: With TF, increasing concentrations of dalteparin significantly prolonged R and K and significantly decreased α and MA. K, α, and MA ratios were significantly different from baseline at all dalteparin concentrations and R was significantly different from baseline at concentrations of 0.625, 1.25, and 2.5 U/mL. With kaolin, only R was significantly different from baseline at dalteparin concentrations of 0.625 and 2.5 U/mL. PiCT detected dalteparin concentrations ≤ 0.625 U/mL, with a good linear correlation (r²=.96, P<.0001). Conclusion: These results suggest that TF‐activated TEG and PiCT assays should be further evaluated as promising new methods for evaluating the effect of LMWH, using doses in the recommended clinical range and prospective clinical studies.     

Effects of storage time on chemistry results from canine whole blood,heparinized whole blood,serum and heparinized plasma

The stability and storage characteristics of 24 blood constituents from dogs including nine enzymes (ALP, ALT, amylase, AST, CK, GGT, GLDH, LDH, lipase), 15 metabolites and minerals (albumin, bile acids, bilirubin, calcium, cholesterol, creatinine, fructosamine, glucose, magnesium, phosphate, potassium, protein, sodium, triglycerides, urea) were studied. Conditions studied included storing of nonanticoagulated and heparinized whole blood for 3 days (Part A), and storing of serum and heparinized plasma for 3 days (Part B). The storage temperature for both studies was +4 degrees C from day 0 to day 1, and +20 degrees C, from day 1 to day 2 and 3. Eight of 24 analytes showed no significant differences (p > 0.05) for three days in whole blood. However, the stability of all 24 analytes greatly improved by storing serum or heparinized plasma compared to nonanticoagulated or heparinized whole blood. In stored serum or heparinized plasma, 20 of 24 analytes showed no significant differences (p < 0.05) for 3 days. Nine of 24 analytes showed significant differences (p < 0.05) between serum and heparinized plasma, where CK, LDH, GGT, and potassium showed differences of possible clinical importance. This study strongly supports the practice of separating serum/plasma from clot/cells as promptly as possible to achieve improved stability for most analytes under test.     

Studies on the role of carbohydrates in the adherence of Malassezia pachydermatis to canine corneocytes in vitro

A panel of four lectins was used to investigate the role of carbohydrates in the adherence of Malassezia pachydermatis to canine corneocytes in vitro . Pretreatment of canine corneocytes with concanavalin A (10 μg ml⁻¹) inhibited ( P < 0.01) the adherence of one out of six M. pachydermatis strains. This effect was abrogated by pre-incubating concanavalin A with its hapten inhibitor (6% methyl α- d -mannopyranoside), suggesting that mannosyl-bearing carbohydrate residues on the epithelial cells serve as ligands for adhesins expressed by this strain. However, treatment of corneocytes with soybean, wheat germ and gorse lectin, and pretreatment of the yeast cells with either of the four lectins had no reproducible effect on the adherence of two strains.     

Assessment of the measurement of canine and feline serum fibroblast growth factor-23 concentrations by automated chemiluminescence immunoassay

This study compared canine and feline fibroblast growth factor (FGF)-23 concentration measurements between automated chemiluminescence assay (CLEIA) and enzyme-linked immunosorbent assay (ELISA). Seventy serum samples each from dogs and cats were evaluated. FGF-23 measurements by CLEIA significantly correlated with those of ELISA in both dogs and cats. The Bland–Altman test showed that FGF-23 between CLEIA and ELISA had fixed and proportional biases, respectively, in both dogs and cats. Measurements by CLEIA were lower than those of ELISA, especially in higher serum FGF-23 concentrations. This study showed that FGF-23 concentrations in dogs and cats can be evaluated by automated CLEIA. However, FGF-23 cannot be directly compared between CLEIA and ELISA.     

Evaluation of a commercial kit for measurement of glycosylated hemoglobin in canine blood

Total glycosylated hemoglobin (HbA(1)) was measured in canine blood samples by conventional macrocolumn ion exchange chromatography and with a commercial glycosylated hemoglobin kit. The two methods correlated well (r= 0.94, p < 0.001) and the reproducibility of the kit method was good. The commercial kit method is recommended for measurement of HbA(1) in canine blood.     

Aggregability and post-transfusion survival of canine platelets in stored whole blood

The effects of whole blood storage time on platelet aggregation and on post-transfusion platelet survival time were assessed in dogs. Citrate phosphate dextrose adenine-1 (CPDA-1) was used as a blood cell preservative. Storage time dependent decay of platelet aggregability was assessed. Platelet aggregation responses to collagen and ADP were maintained for at least 8 hr at room temperature. During blood storage, immunoglobulin became nonspecifically bound to platelets, suggesting the potential for immune destruction of platelets by the mononuclear phagocyte system after transfusion. To assess this assumption, the survival times of infused platelets, which were stored for 0 to 8 hr in whole blood, were measured. Post-transfusion survival of platelets was not affected by these storage times. These results suggest that canine platelets maintain viability when stored at room temperature for up to 8 hr in CPDA-1 treated whole blood intended for transfusion.     

Evaluation of a hematology analyzer with canine and feline blood

A semiautomatic electronic blood cell counter (Sysmex F-800:Toa Medical Electronics Europa Gmbh, Hamburg, Germany) was evaluated using canine and feline blood, following the International Committee for Standardization in Hematology protocol (ICSH, 1984). Precision and overall reproducibility were acceptable for all the parameters studied except for the feline platelet count, in which overlapping of erythrocyte and platelet populations prohibited determination of an accurate platelet count. Since carry-over from canine hematocrit values and platelet counts and from feline hematocrit values was unsatisfactory, the use of a blank diluent sample between different analyses was necessary. Linearity of the analyzer was acceptable in the studied range. Thirty canine and feline blood samples were analyzed using the Sysmex F-800 and a manual method. Correlations between both methods were acceptable for all the parameters, except for feline platelet count and erythrocyte indices for both species. In the storage study, red blood cell count and hemoglobin concentration were the parameters with the longest stability (72 hours at 4 degrees C and 25 degrees C) in both species. A statistically significant increase in MCV was obtained at 12 hours post-extraction in canine samples stored at 25 degrees C and at 24 hours in refrigerated samples. Feline leucocyte counts showed a downward trend at 12 hours post-extraction at both temperatures. Canine platelet count decreased significantly at 6 hours post-extraction in samples stored at 4 degrees C. During the evaluation period, Sysmex F-800 was user friendly and appeared well suited for routine canine and feline blood cell analysis.     

Artifactual changes in canine blood following storage, detected using the ADVIA 120 hematology analyzer

BACKGROUND: Artifactual changes in blood may occur as a consequence of delayed analysis and may complicate interpretation of CBC data. OBJECTIVE: The aim of this study was to characterize artifactual changes in canine blood, due to storage, using the ADVIA 120 hematology analyzer. METHODS: Blood samples were collected into EDTA from 5 clinically healthy dogs. Within 1 hour after blood sample collection and at 12, 24, 36 and 48 hours after storage of the samples at either 4 degrees C or room temperature (approximately 24 degrees C), a CBC was done using the ADVIA 120 and multispecies software. A linear mixed model was used to statistically evaluate significant differences in values over time, compared with initial values. RESULTS: The HCT and MCV were increased significantly after 12 hours of collection at both 4 degrees C and 24 degrees C, and continued to increase through 48 hours. The MCHC initially decreased significantly at 12-24 hours and then continued to decrease through 48 hours at both temperatures. Changes in HCT, MCV, and MCHC were greater at 24 degrees C than at 4 degrees C at all time points. A significant increase in MPV and a decrease in mean platelet component concentration were observed at all time points at 24 degrees C. Samples stored at 24 degrees C for 48 hours had significantly higher percentages of normocytic-hypochromic RBCs, and macrocytic-normochromic RBCs, and lower platelet and total WBC counts. CONCLUSIONS: Delayed analysis of canine blood samples produces artifactual changes in CBC results, mainly in RBC morphology and platelet parameters, that are readily detected using the ADVIA 120. Refrigeration of specimens, even after 24 hours of storage at room temperature, is recommended to improve the accuracy of CBC results for canine blood samples.