Stability of coagulation factors in feline fresh frozen plasma intended for transfusion after 1 year of storage.

OBJECTIVES: The most common use of plasma transfusion is for haemostatic purposes, but coagulation factor activities in stored feline plasma are unknown. The concentration and stability of coagulation factors I (fibrinogen), II, V, VII, VIII, IX, X, XI and XII in feline fresh frozen plasma (fFFP) stored for 1 year were studied. METHODS: Fifty-five units of fFFP were produced from 55 fresh whole-blood donations obtained from indoor healthy blood donor cats. Twenty-one units were stored for <2 weeks (T0) and 34 were stored for 1 year (T1). After the completion of storage, specific coagulation factor activities for factors II, V, VII, VIII, IX, X, XI and XII were tested using modified one-stage activated partial thromboplastin or prothrombin time assays. Fibrinogen was determined using the Clauss method. RESULTS: Significantly decreased activities were observed for factors II (T0: 101.94% ± 19.06%; T1: 73.23% ± 39.06% [P = 0.001]), VII (T0: 102.78% ± 24.69%; T1: 60.08% ± 38.17% [P <0.001]), VIII (T0: 77.52% ± 30.39%; T1: 50.32% ± 23.8% [P = 0.001]), XI (T0: 88.76% ± 22.73%; T1: 66.28% ± 22.2% [P = 0.001]) and XII (T0: 89.50% ± 21.85%; T1: 55.46% ± 23.18% [P <0.001]) when comparing units at time 0 and after 1 year of storage. No significant difference was observed for factors IX (T0: 84.86% ± 29.35%; T1: 71.37% ± 22.23% [P = 0.064]) and X (T0: 96.24% ± 25.1%; T1: 83.91% ± 49.54% [P = 0.236]). Unexpectedly, a significant increase was observed for factor V (T0: 71.94% ± 24.14%; T1: 97.89% ± 62.33%; P = 0.046). Fibrinogen was 2.76 ± 1.09 g/l at T1. Factors VIII, XII and VII had the lowest mean activities after 1 year. CONCLUSIONS AND RELEVANCE: Although a decrease in most coagulation factors activities was noted with storage, 1-year-old fFFP was haemostatically active in vitro. The most suitable factors for quality control assessment of fFFP are factors VII and VIII. Approximately 13-20 ml/kg of fFFP is required to administer a minimum of 10 IU/kg coagulation factor activity.

Quantitative assessment of infusion pump-mediated haemolysis in feline packed red blood cell transfusions.

OBJECTIVES: Haemolysis caused by the use of peristaltic infusion pumps (PIPs) has been described in human and canine packed red blood cells (pRBCs). The aim of this study was to evaluate the effects of two different linear PIPs on the haemolysis of feline pRBC units stored for a long time. METHODS: Feline pRBC units stored with adenine, dextrose, mannitol and sodium chloride (SAGM) were manufactured. After 35-42 days of storage at 2-4°C, a line administration system with a 180 µm filter was attached to every pRBC bag, the system was drained by gravity alone (8 drops/min) and a 1.3 ml sample was collected (G). A NIKI V4 pump was then used at a flow rate of 25 ml/h, the flow was stopped when the infusion system was filled with blood coming from the infusion pump and another 1.3 ml sample was collected (NK). Finally, an Infusomat FmS pump was evaluated, collecting another 1.3 ml sample (IM). Packed cell volume (PCV) was measured in all samples by microhaematocrit centrifugation, total haemoglobin (HGB) was measured using a specific haemoglobin analyser and, after centrifugation, free HGB was determined by spectrophotometry. The percentage of haemolysis was calculated. Friedman's test was used to compare the samples. RESULTS: Fifteen feline pRBC units were evaluated. The average degree of haemolysis for sample G (gravity-assisted) was 1.12%. Comparison of the degree of gravity-assisted haemolysis with haemolysis in PIP NK (1.13%) and IM (1.14%) samples revealed no significant differences, with differences of only 0.01% and 0.02%, respectively. CONCLUSIONS AND RELEVANCE: The results of this study demonstrate that the use of two common PIPs in veterinary hospitals does not produce levels of haemolysis that are significantly different than that caused by gravity alone during transfusion of feline pRBCs at a rate of 25 ml/h.

Adiponectin as a sepsis biomarker in dogs: Diagnostic and prognostic value.

BACKGROUND: Adiponectin (ADPN) is an adipocytokine with insulin-sensitizing, vascular-protective, and anti-inflammatory properties for which concentration changes occur in response to inflammation. Little is known about the regulation of ADPN and the impact of this adipocytokine in septic dogs. OBJECTIVE: We aimed to assess the diagnostic and prognostic value of ADPN vs other traditional acute-phase proteins (APPs), such as albumin (ALB), haptoglobin (HPT), fibrinogen (FBG), ferritin (FRT), and C-reactive protein (CRP) in dogs with naturally acquired sepsis. METHODS: This prospective observational study included 20 dogs with sepsis, 27 with low-grade systemic inflammation (LGSI), and 18 clinically healthy dogs as controls. For method analyses, plasma samples were obtained from all dogs on admission and then every 24-48 hours until discharge or death in the septic group. RESULTS: Septic dogs had lower ADPN (2.4 ± 0.46 vs 4.5 ± 0.41mg/L, P < .001) dand ALB (17 ± 1 vs 22 ± 0.8g/L, P = .002), and tended to have higher CRP (87 ± 4.8 vs 73 ± 4.1mg/L, P < .079) concentrations than dogs with LGSI on admission. Only ADPN and ALB were able to successfully discriminate animals with LGSI from those presenting with sepsis with areas under the curve (AUCs) for the receiver operating characteristic (ROC) curves of 0.811 and 0.789, respectively. In the septic group, ADPN concentration did not differ between survivors and non-survivors, either on admission or at discharge or death. CONCLUSIONS: Although plasma ADPN can be used as a reliable negative APP in dogs with sepsis, further studies are warranted to confirm the usefulness of this biomarker in terms of disease progression and recovery.

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.

Long-Term Efficacy and Safety of Insulin and Glucokinase Gene Therapy for Diabetes: 8-Year Follow-Up in Dogs.

Diabetes is a complex metabolic disease that exposes patients to the deleterious effects of hyperglycemia on various organs. Achievement of normoglycemia with exogenous insulin treatment requires the use of high doses of hormone, which increases the risk of life-threatening hypoglycemic episodes. We developed a gene therapy approach to control diabetic hyperglycemia based on co-expression of the insulin and glucokinase genes in skeletal muscle. Previous studies proved the feasibility of gene delivery to large diabetic animals with adeno-associated viral (AAV) vectors. Here, we report the long-term (∼8 years) follow-up after a single administration of therapeutic vectors to diabetic dogs. Successful, multi-year control of glycemia was achieved without the need of supplementation with exogenous insulin. Metabolic correction was demonstrated through normalization of serum levels of fructosamine, triglycerides, and cholesterol and remarkable improvement in the response to an oral glucose challenge. The persistence of vector genomes and therapeutic transgene expression years after vector delivery was documented in multiple samples from treated muscles, which showed normal morphology. Thus, this study demonstrates the long-term efficacy and safety of insulin and glucokinase gene transfer in large animals and especially the ability of the system to respond to the changes in metabolic needs as animals grow older.

Plasma iron, C-reactive protein, albumin, and plasma fibrinogen concentrations in dogs with systemic inflammatory response syndrome.

OBJECTIVE: To investigate the diagnostic and prognostic value over time of plasma iron compared with the inflammatory markers albumin, C-reactive protein (CRP), and fibrinogen in dogs with systemic inflammatory response syndrome (SIRS). DESIGN: Prospective observational study of sequentially enrolled dogs. SETTING: ICU of a veterinary teaching hospital. ANIMALS: One hundred and sixteen client-owned dogs: 54 dogs with SIRS or sepsis, 42 with focal inflammation, and 20 clinically healthy dogs. MEASUREMENTS AND MAIN RESULTS: Blood samples were obtained on admission in all study groups, and then on alternate days until discharge or death in both inflammation groups. On admission, dogs with SIRS had significantly lower plasma iron (65 ± 5.8 µg/dL, P = 0.001) concentrations than dogs with focal inflammation (89.5 ± 6.2 µg/dL, P = 0.001). Plasma iron, albumin, and CRP effectively discriminated the SIRS/sepsis group from those presenting with focal inflammation with areas under the curve for the receiver operating curves of 0.679, 0.834, and 0.704, respectively. The admission values for these variables did not discriminate survivors from nonsurvivors within the SIRS/sepsis group. However, the magnitude of increase in iron concentration and the decrease in CRP concentration from admission to hospital discharge was higher in survivors than in nonsurvivors within the SIRS/septic group (22.8 vs. 2.51 µg/dL, respectively, P = 0.021 for iron; -67.1 vs. -4.1 mg/L, respectively, P = 0.002 for CRP), resulting in iron and CRP concentrations at hospital discharge for survivors similar to those in the focal inflammation group. CONCLUSION: Hypoferremia is a sensitive marker of systemic inflammation in dogs. In this study, the increase in iron concentrations during the hospitalization period of SIRS/septic dogs was associated with a better prognosis, suggesting that plasma iron in combination with CRP and albumin concentrations might be used to monitor dogs with inflammatory disease processes.

Treatment of diabetes and long-term survival after insulin and glucokinase gene therapy.

Diabetes is associated with severe secondary complications, largely caused by poor glycemic control. Treatment with exogenous insulin fails to prevent these complications completely, leading to significant morbidity and mortality. We previously demonstrated that it is possible to generate a "glucose sensor" in skeletal muscle through coexpression of glucokinase and insulin, increasing glucose uptake and correcting hyperglycemia in diabetic mice. Here, we demonstrate long-term efficacy of this approach in a large animal model of diabetes. A one-time intramuscular administration of adeno-associated viral vectors of serotype 1 encoding for glucokinase and insulin in diabetic dogs resulted in normalization of fasting glycemia, accelerated disposal of glucose after oral challenge, and no episodes of hypoglycemia during exercise for >4 years after gene transfer. This was associated with recovery of body weight, reduced glycosylated plasma proteins levels, and long-term survival without secondary complications. Conversely, exogenous insulin or gene transfer for insulin or glucokinase alone failed to achieve complete correction of diabetes, indicating that the synergistic action of insulin and glucokinase is needed for full therapeutic effect. This study provides the first proof-of-concept in a large animal model for a gene transfer approach to treat diabetes.

Severe life-threatening hypokalemia in a cat with suspected distal renal tubular acidosis.

OBJECTIVE: Description of the clinical presentation and management of a critically ill cat with profound hypokalemia associated with a suspicion of distal renal tubular acidosis (DRTA) and secondary hyperaldosteronism. CASE SUMMARY: A cat was presented with severe generalized weakness and acute ventilatory failure associated with severe hypokalemia. The acid-base analysis and complete analytical profile of the urine confirmed the presence of a normal anion-gap metabolic acidosis with a urine pH of 7, a disorder consistent with DRTA. The high plasma renin activity, high aldosterone concentration, and low normal plasma aldosterone concentration/plasma renin activity ratio suggested secondary hyperaldosteronism. The management of the patient in the ICU was successful. No identifiable cause could be determined as a cause for the DRTA, so the disorder was assumed to be the primary problem. NEW OR UNIQUE INFORMATION PROVIDED: DRTA is a rare disorder occasionally reported in the veterinary literature; it is especially rare in cats. Complete diagnostic evaluation was necessary to identify the reported disorders as the cause of the clinical presentation. To the author's knowledge, this is the first case reporting DRTA, and a simultaneously documented mineralocorticoid response, as a cause of a life-threatening hypokalemia.

Effects of sedation with midazolam and butorphanol on resistive and pulsatility indices in healthy dogs.

Resistive index (RI) and pulsatility index (PI) are indirect measurements of blood flow resistance that may be measured by pulsed wave Doppler ultrasonography. Chemical restrain may potentially alter the indices although it is required to perform ultrasonography in some patients. The purpose of this study was to describe values for both intrarenal and ocular RI and PI within the same subject in clinically normal dogs sedated with a midazolam and butorphanol combination and evaluate if there are any significant changes between sedated and nonsedated dogs. Fifteen healthy Beagle dogs were studied by Duplex Doppler interrogation in interlobar or arcuate arteries of the kidney and long posterior ciliary artery. Pulse rate and systolic blood pressure were also determined. All measurements were recorded before and after the administration of a sedative combination of midazolam (0.2 mg/kg) and butorphanol (0.2 mg/kg). Mean comparison tests (paired t-tests or Wilcoxon's rank-sum test) were used to determine if any significant differences existed between right and left renal values or right and left ocular values. A correlation study (Pearson or Spearman) was applied between RI, PI, and systolic pressure, and pulse rate. RI and PI were significantly higher in sedated Beagles than in unsedated Beagles. There was neither correlation between index and systolic blood pressure nor pulse rate. In conclusion, provided that normal RI and PI increase in sedated animals, then reference ranges should be higher when sedated--healthy or ill--animals are evaluated.

Doppler ultrasonographic estimation of renal and ocular resistive and pulsatility indices in normal dogs and cats.

Resistive index (RI) and pulsatility index (PI) are indirect measurements of blood flow resistance that may be used to evaluate vascular changes in renal and ophthalmologic diseases. To our knowledge, no reports are available describing values for renal and ocular PI index in the unsedated dog and ocular RI and PI indices in the unsedated cat. The purpose of this study was to measure normal values for both intrarenal and ocular RI and PI within the same subject in unsedated clinically normal dogs and cats. Twenty-seven dogs and 10 cats were considered healthy by means of physical examination, CBC, biochemical profile, urinalysis, and ultrasonography. Systolic blood pressure was measured by Doppler ultrasonography. Intrarenal and ocular arteries were scanned by pulsed Doppler ultrasonography to calculate RI and PI. No significant differences were noted between the values obtained for the right vs. the left kidney and eye. The upper values of these indices were calculated as mean + 2 standard deviations resulting in 0.72 and 1.52 for dog renal RI and PI; 0.7 and 1.29 for cat renal RI and PI; 0.76 and 1.68 for dog ocular RI and PI; and 0.72 and 1.02 for cat ocular RI and PI.

Clinico-pathological findings and coagulation disorders in 45 cases of canine babesiosis in Spain.

A retrospective study of clinical cases of babesiosis in dogs examined at the Veterinary Teaching Hospital, Rof Codina, from January 2003 to October 2004 is presented. The diagnosis was confirmed by direct observation of large piroplasms in stained blood smears. Dogs with concurrent diseases were excluded from the study. Clinical signs, complete blood count, serum biochemistry and hemostasis profiles were obtained. The observed clinical signs were due to hemolytic anemia and inflammatory responses but the most relevant clinico-pathological findings were related to alterations in hemostasis. All dogs presented with thrombocytopenia and 20% had disseminated intravascular coagulation syndrome. Anemia of variable severity was observed in most of the dogs.

Effects of preoperative administration of meloxicam on whole blood platelet aggregation, buccal mucosal bleeding time, and haematological indices in dogs undergoing elective ovariohysterectomy.

The purpose of the study was to evaluate the effect of preoperative administration of meloxicam, a non-steroidal anti-inflammatory drug used for pain control, on primary haemostasis in dogs. Twenty healthy female dogs undergoing elective ovariohysterectomy were enrolled in the study. Sixty minutes before pre-anaesthesia, a single dose of meloxicam (0.2 mg/kg) was randomly administered intravenously (IV) to 10 dogs (treatment group) while control dogs received an equivalent volume of saline solution IV. Platelet aggregation, buccal mucosa bleeding time, platelet count and haematological indices were measured at 0, 1, 6 and 24 h after administration of meloxicam. Since significant differences between groups were not observed for any of the measured parameters, preoperative administration of meloxicam may be used for pain control before elective ovariohysterectomy in healthy dogs, without compromising primary haemostasis.