Evaluation of bacterial growth, effects on albumin, and coagulation factors in canine fresh frozen plasma administered as continuous rate infusion exposed to room temperature for 12 hours.

OBJECTIVES: To determine the risk of bacterial growth and to analyze the stability of albumin and coagulation factors in canine fresh frozen plasma (FFP) units exposed to room temperature (24°C) administered as a continuous rate infusion (CRI) for 12 hours. DESIGN: Ex vivo study. SETTING: University teaching hospital and pet blood bank. ANIMALS: None. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: An FFP CRI was simulated to replicate the standard routine procedure used in dogs. Plasma samples were collected before starting the CRI (H0), after 4 hours (H4), and after 12 hours (H12). Bacterial culture of FFP was performed and albumin concentration and specific activity levels for factors V, VII, VIII, and IX were measured and compared. All plasma culture results were negative. There were no statistically significant differences at any time point in the factor VIII activity (median 105.5% [range, 75.6%-142.0%] at H0; median 107.8% [range, 75.0%-172.7%] at H4; and median 112.1% [range, 81.7%-171.0%] at H12); factor IX activity (median 119.3% [range, 89.1%-175.9%] at H0; median 123.1% [range, 72.5%-172.7%] at H4; and median 118.3% [range, 86.6%-177.5%] at H12); or albumin concentration (median 21.0 g/L [range, 17.0-23.0 g/L] at H0 and median 20.0 g/L [range, 17.0-24.0 g/L] at H12). A slight but significant increase in factor V activity was observed when comparing H0 (median 107.0% [range, 71.0%-159.0%]) to H4 (median 117.7% [range, 71.0%-176.7%]) (P = 0.002) or H12 (median 116.2% [range, 71.0%-191.6%]) (P = 0.001). A slight but significant increase in factor VII activity was observed when comparing H0 (median 115.4% [range, 70.6%-183.7%]) to H4 (median 118.2% [range, 82.7%-194.6%]) (P = 0.005); H0 to H12 (median 128.7% [range, 86.4%-200.0%]) (P < 0.001); and H4 to H12 (P = 0.002). CONCLUSIONS: FFP CRI at room temperature for 12 hours could be considered safe with regard to risk for bacterial growth and also effective by providing albumin and clotting factors.

In vitro hemolysis of stored units of canine packed red blood cells.

BACKGROUND: Hemolysis is an important quality parameter of packed red blood cells (pRBCs) that is used to assess the cellular integrity of stored blood units. According to human standards, hemolysis at the end of storage must not exceed 1%, as otherwise it may be responsible for decreased transfusion effectiveness and acute life-threatening reactions. OBJECTIVES: This prospective study was designed to evaluate the hemolysis of canine pRBCs stored in an additive solution containing adenine, dextrose, mannitol, and sodium chloride, and to assess its associations with storage time, duration of the collection process, collection disturbances, and with the final volume and PCV of the pRBCs units. METHODS: One hundred eighty pRBCs units were collected from canine donors. Hemolysis of the pRBCs units was determined immediately after processing (t = 0). The units were then stored and retested (t = 1) either before administration (during weeks 2, 3, 4, 5, or 6 of storage) or at the end of the storage period (42 d) if not used. RESULTS: Mean hemolysis at t = 0 was 0.09% (SD 0.06) and increased during storage, at a more pronounced rate from the 5th (mean values of 0.52%, SD 0.29) to the 6th week (1.2%, SD 0.72). Almost 51% of the units with 36-42 days of shelf-life showed more than 1% hemolysis. Disturbances in the collection process, the volume of the whole blood units, and the volume of stored pRBCs units or their PCV were not related to pRBCs hemolysis. CONCLUSIONS: According to human blood bank recommendations regarding acceptable hemolysis, canine pRBCs stored for more than 35 days should be tested to ensure <1% hemolysis prior to administration.

In vitro quality control analysis after processing and during storage of feline packed red blood cells units.

BACKGROUND: During the storage of packed red blood cells (pRBC), packed cell volume (PCV), bacterial contamination and percentage of haemolysis [percentage of free haemoglobin (HGB) in relation to the total HGB] are important quality parameters. Both PCV and haemolysis are indicators of the cellular integrity of stored units. There are no published experimental studies that evaluated these parameters during storage of feline pRBC using SAGM (adenine, dextrose, mannitol and sodium chloride) as the additive solution. The present study aims to (1) evaluate the quality of feline pRBCs stored in SAGM; (2) test for the semi-closed system's suitability for use and risk of bacterial contamination; (3) establish the maximum storage time that may be appropriate to meet the criteria established by the United States Food and Drug Administration (US-FDA) guidelines for human blood banking; and (4) evaluate the need to calculate the percentage of haemolysis prior to the administration of units stored for more than 4 weeks. Four hundred eighty nine feline pRBC units were analyzed. Bacterial culture, PCV and percentage of haemolysis were determined within 6 h after processing (t0). One hundred and eighty units were re-tested for haemolysis and PCV after 29-35 days of storage (t1) and 118 units after 36-42 days (t2). RESULTS: Bacterial contamination was not detected in any pRBC unit. Mean PCV at t0 was 52.25% (SD: ±5.27) and decreased significantly (p < 0.001) during storage to 48.15% (SD: ±3.79) at t1 and to 49.34% (SD: ±4.45) at t2. Mean percentage of haemolysis at t0 was 0.07% (SD: ±0.06) and increased significantly (p < 0.001) to 0.69% (SD: ±0.40) at t1 and to 0.81% (SD: ±0.47) at t2. In addition, 13.88% and 19.49% of pRBC units exceeded 1% haemolysis at t1 and t2, respectively. CONCLUSIONS: According to the US-FDA guidelines for human blood banking that recommend a maximum of 1% haemolysis, the results of this study show that all feline pRBC units with less than 24 h of shelf life have low levels of haemolysis. However, units preserved up to 28 days can only be administered if tested for haemolysis before use, since 13.88% units exceeded the 1% limit. The semi-closed system was considered safe for use as bacterial contamination was not detected in any pRBC unit.

Characterization of α-, ß- and p120-Catenin Expression in Feline Mammary Tissues and their Relation with E- and P-Cadherin.

BACKGROUND: Abnormal catenin expression has been related to mammary carcinogenesis in both human and canine species and they are considered tumor- and invasion-suppressor molecules; however, in feline mammary tissues they have been scarcely studied. MATERIALS AND METHODS: The immunohistochemical expression of α-, ß- and p120-catenin was studied in a series of normal feline mammary glands, hyperplastic/dysplastic lesions and benign and malignant mammary tumors. Their relationship with clinicopathological parameters and with E- and P-cadherin expression was assessed. RESULTS: Normal tissues, hyperplastic/dysplastic lesions and benign tumors expressed α-, ß- and p120-catenin in the membrane of more than 75% of the luminal epithelial cells, while in malignant tumors, there was a reduction in their membranous expression and a p120-catenin cytoplasmic expression in 40%. Reduced α-catenin expression was related to tumor features with prognostic value, namely tumor size (p=0.0203) and necrosis (p=0.0205). The expression of α-, ß- and p120-catenin were individually related to each other and collectively associated with E-cadherin expression. CONCLUSION: The results demonstrate a relationship between feline mammary carcinogenesis and decreased expression of catenins, suggesting that they may represent a valuable tool in the diagnosis of feline mammary neoplasms.

Volume-dependent hemodynamic effects of blood collection in canine donors - evaluation of 13% and 15% of total blood volume depletion.

BACKGROUND: There is no consensus regarding the blood volume that could be safely donated by dogs, ranging from 11 to 25% of its total blood volume (TBV). No previous studies evaluated sedated donors. AIM: To evaluate the hemodynamic effects of blood collection from sedated and non-sedated dogs and to understand if such effects were volume-dependent. MATERIALS AND METHODS: Fifty three donations of 13% of TBV and 20 donations of 15% TBV were performed in dogs sedated with diazepam and ketamine. Additionally, a total of 30 collections of 13% TBV and 20 collections of 15% TBV were performed in non-sedated dogs. Non-invasive arterial blood pressures and pulse rates were registered before and 15 min after donation. RESULTS: Post-donation pulse rates increased significantly in both sedated groups, with higher differences in the 15% TBV collections. Systolic arterial pressures decreased significantly in these groups, while diastolic pressures increased significantly in 13% TBV donations. Non-sedated groups revealed a slight, but significant, SBP decrease. No clinical signs related to donations were registered. CONCLUSION: These results suggest that the collection of 15% TBV in sedated donors induces hemodynamic variations that may compromise the harmlessness of the procedure, while it seems to be a safe procedure in non-sedated dogs.

Aberrant P-cadherin expression is associated to aggressive feline mammary carcinomas.

BACKGROUND: Cadherins are calcium-dependent cell-to-cell adhesion glycoproteins playing a critical role in the formation and maintenance of normal tissue architecture. In normal mammary gland, E-cadherin is expressed by luminal epithelial cells, while P-cadherin is restricted to myoepithelial cells. Changes in the expression of classical E- and P-cadherins have been observed in mammary lesions and related to mammary carcinogenesis. P-cadherin and E-cadherin expressions were studied in a series of feline normal mammary glands, hyperplastic/dysplastic lesions, benign and malignant tumours by immunohistochemistry and double-label immunofluorescence. RESULTS: In normal tissue and in the majority of hyperplastic/dysplastic lesions and benign tumours, P-cadherin was restricted to myoepithelial cells, while 80% of the malignant tumours expressed P-cadherin in luminal epithelial cells. P-cadherin expression was significantly related to high histological grade of carcinomas (p <0.0001), tumour necrosis (p = 0.001), infiltrative growth (p = 0.0051), and presence of neoplastic emboli (p = 0.0401). Moreover, P-cadherin positive carcinomas had an eightfold likelihood of developing neoplastic emboli than negative tumours. Cadherins expression profile in high grade and in infiltrative tumours was similar, the majority expressing P-cadherin, regardless of E-cadherin expression status. The two cadherins were found to be co-expressed in carcinomas with aberrant P-cadherin expression and preserved E-cadherin. CONCLUSIONS: The results demonstrate a relationship between P-cadherin expression and aggressive biological behaviour of feline mammary carcinomas, suggesting that P-cadherin may be considered an indicator of poor prognosis in this animal species. Moreover, it indicates that, in queens, the aberrant expression of P-cadherin is a better marker of mammary carcinomas aggressive behaviour than the reduction of E-cadherin expression. Further investigation with follow-up studies in feline species should be conducted in order to evaluate the prognostic value of P-cadherin expression in E-cadherin positive carcinomas.

Prevalence of antimicrobial resistance in enteric Escherichia coli from domestic pets and assessment of associated risk markers using a generalized linear mixed model.

Antimicrobial resistance (AMR) is a growing global public health problem, which is caused by the use of antimicrobials in both human and animal medical practice. The objectives of the present cross-sectional study were as follows: (1) to determine the prevalence of resistance in Escherichia coli isolated from the feces of pets from the Porto region of Portugal against 19 antimicrobial agents and (2) to assess the individual, clinical and environmental characteristics associated with each pet as risk markers for the AMR of the E. coli isolates. From September 2009 to May 2012, rectal swabs were collected from pets selected using a systematic random procedure from the ordinary population of animals attending the Veterinary Hospital of Porto University. A total of 78 dogs and 22 cats were sampled with the objective of isolating E. coli. The animals' owners, who allowed the collection of fecal samples from their pets, answered a questionnaire to collect information about the markers that could influence the AMR of the enteric E. coli. Chromocult tryptone bile X-glucuronide agar was used for E. coli isolation, and the disk diffusion method was used to determine the antimicrobial susceptibility. The data were analyzed using a multilevel, univariable and multivariable generalized linear mixed model (GLMM). Several (49.7%) of the 396 isolates obtained in this study were multidrug-resistant. The E. coli isolates exhibited resistance to the antimicrobial agent's ampicillin (51.3%), cephalothin (46.7%), tetracycline (45.2%) and streptomycin (43.4%). Previous quinolone treatment was the main risk marker for the presence of AMR for 12 (ampicillin, cephalothin, ceftazidime, cefotaxime, nalidixic acid, ciprofloxacin, gentamicin, tetracycline, streptomycin, chloramphenicol, trimethoprim-sulfamethoxazole and aztreonam) of the 15 antimicrobials assessed. Coprophagic habits were also positively associated with an increased risk of AMR for six drugs, ampicillin, amoxicillin-clavulanic acid, cephamycin, ciprofloxacin, streptomycin, and trimethoprim-sulfamethoxazole. In summary, pets with a record of one or more previous quinolone treatments and exhibiting coprophagic habits were at an increased risk of harboring multidrug-resistant E. coli strains in their feces compared to pets without these characteristics. AMR is a serious global problem, and assessing the risk markers for the presence of drug-resistant bacteria in pets, a very close source of resistance determinants to humans, is essential for the implementation of safe handling procedures for companion animals and for the prudent selection of antimicrobial compounds in veterinary practice.

Effects of repeated blood donations on iron status and hematologic variables of canine blood donors.

OBJECTIVE: To evaluate the bone marrow regenerative response and iron status of canine blood donors subjected to repeated blood collections for 1 year. DESIGN: Prospective cohort study. ANIMALS: 57 blood donor dogs. PROCEDURES: Hematologic variables, including reticulocyte percentage, were evaluated before and 10 days after each blood collection in 16 dogs donating 13% of total blood volume (TBV) every 2 months (group 1), 16 dogs donating 13% of TBV every 3 months (group 2), and 25 dogs donating 15% of TBV every 3 months (group 3) for 1 year. Serum concentrations of iron, transferrin, and ferritin were analyzed before inclusion in the study and 10 days after the last donation. RESULTS: Significant increases in RBC distribution width, platelet count, WBC count, and reticulocyte percentage were detected after blood donation in all groups. Dogs of group 2 had a significantly higher serum ferritin concentration than did dogs of group 1; dogs of group 1 had a significant decrease in serum ferritin concentration. A positive correlation between the number of blood donations and both RBC distribution width and reticulocyte percentage was found for all groups. CONCLUSIONS AND CLINICAL RELEVANCE: All blood donation regimens induced a bone marrow regenerative response, which was able to restore depleted blood cells within 10 days after blood donation while maintaining iron status within the calculated reference range. However, dogs donating 13% of TBV every 2 months had a significant decrease in iron stores, which suggested that iron-related variables must be monitored during prolonged blood donor programs.

Matrix metalloproteinase-9 expression in mammary gland tumors in dogs and its relationship with prognostic factors and patient outcome.

OBJECTIVE: To immunohistochemically evaluate matrix metalloproteinase (MMP)-9 expression in benign and malignant mammary gland tumors (MMTs) in dogs and relate expression to prognostic factors and patient outcome. ANIMALS: 118 female dogs with naturally occurring mammary gland tumors and 8 dogs without mammary gland tumors. PROCEDURES: 24 benign mammary gland tumors and 94 MMTs (1/affected dog) were obtained during surgical treatment; control mammary gland tissue samples were collected from unaffected dogs after euthanasia for reasons unrelated to the study. Tumors were evaluated for proliferation, invasive growth, histologic grade, and metastatic capacity; expression of MMP-9 was determined immunohistochemically, and its relationship with clinical and histologic findings was investigated. For dogs with MMTs, follow-up continued for 2 years; data were used to compute overall survival time and disease-free interval and construct survival curves. RESULTS: MMTs had significantly higher MMP-9 expression in stromal cells and in neo-plastic cells than did the benign neoplasms. Stromal MMP-9 expression was also higher in highly proliferative tumors and in tumors with invasive growth, high histologic grade, and metastatic capacity. Furthermore, tumors from patients with shorter overall survival times and disease-free intervals had higher expression of MMP-9 in stromal cells. CONCLUSIONS AND CLINICAL RELEVANCE: In dogs with MMTs, level of MMP-9 expression by stromal cells was related to factors of poor prognosis and shorter overall survival times and disease-free intervals. These results suggested that MMP-9 produced by tumor-adjacent stromal cells contributed to MMT progression in female dogs and that assessment of MMP-9 expression may be a valuable prognostic factor.