Update on Canine and Feline Blood Donor Screening for Blood-Borne Pathogens.

An update on the 2005 American College of Veterinary Internal Medicine (ACVIM) Consensus Statement on blood donor infectious disease screening was presented at the 2015 ACVIM Forum in Indianapolis, Indiana, followed by panel and audience discussion. The updated consensus statement is presented below. The consensus statement aims to provide guidance on appropriate blood-borne pathogen testing for canine and feline blood donors in North America.

Prolonged bleeding time of Chediak-Higashi cats corrected by platelet transfusion.

Cats with the Chediak-Higashi syndrome (CHS) have a platelet storage pool deficiency (SPD). Ten CHS cats were transfused with a concentrate of 51Cr-labeled platelets prepared from normal donor cats. One hour after transfusion, the donor platelet count in CHS recipient cats was 40,000-60,000/microliters. Bleeding time before transfusion was 9.1 +/- 3.0 min. When donor platelet count in CHS cats was 50,000/microliters, bleeding time was 1.7 +/- 0.2 min. Bleeding time of normal cats was 1.4 +/- 0.3 min. Bleeding time increased to 3.3 +/- 0.2 min and to 5.3 +/- 0.2 min when the platelet count was 30,000/microliters, and 15,000/microliters, respectively. The close inverse relationship between bleeding time and number of donor platelets in CHS cats (r = -0.92), suggests that prolonged bleeding time is due to a platelet abnormality, that platelet transfusion can effectively correct prolonged bleeding time in an animal model of platelet SPD and that CHS cats may be an appropriate animal model to evaluate hemostatic capabilities of transfused platelets.

von Willebrand factor is present in the vascular endothelium from normal dogs and from Doberman pinscher dogs with a plasma von Willebrand factor deficiency.

An immunohistochemical study was undertaken to determine the presence and distribution of von Willebrand factor antigen (vWf:Ag) in blood vessels from normal dogs and from Doberman pinscher dogs with a marked plasma deficiency of vWf. vWf:Ag could not be detected in plasma from the Doberman pinscher dogs by ristocetin- and botrocetin-induced platelet agglutination or by EIA. An ELISA assay revealed vWf:Ag levels that were between 2-4% of that in normal canine plasma. Factor VIII:C activity was 30-46% of normal. The activated partial thromboplastin time (APTT) was increased but not the one-stage prothrombin time (OSPT). Four different antibody preparations were used in this study to detect vWf--a monoclonal and a polyclonal antibody prepared against human vWf and 2 polyclonal antibodies against canine vWf. vWf:Ag was detected with monospecific antibody in endothelial cells in veins, venules, and arterioles from normal dogs and vWf-deficient dogs. The histofluorescence observed in vessels of vWf-deficient dogs was indistinguishable from that observed in vessels from normal dogs.

Effect of exercise, DDAVP, and epinephrine on the factor VIII:C/von Willebrand factor complex in normal dogs and von Willebrand factor deficient Doberman pinscher dogs.

Endothelial cells in biopsied blood vessels from von Willebrand factor (vWf)-deficient Doberman pinscher dogs contain immunologically detectable vWf. These dogs and normal dogs were treated with DDAVP (0.6 microgram/kg) and epinephrine (0.5 microgram/kg/min for 30 minutes) and were exercised, using 5 different exercise protocols, (3-4 m/s for 5-40 minutes at 0-5% grade) to determine if treatments reported to increase plasma factor VIII:C/vWf complex in humans would elevate canine plasma vWf. Following the two most strenuous exercise conditions--30 and 40 minutes--plasma von Willebrand factor antigen (vWf:Ag) increased in normal dogs by 30% and 70%, respectively. Factor VIII:C was increased 47% by the most strenuous exercise conditions. The vWf-deficient dogs would not exercise beyond 30 minutes and neither vWf:Ag nor factor VIII:C activity increased. Following DDAVP, plasma vWf:Ag increased in the normal dogs by 47% and factor VIII:C activity was increased by 48%. Factor VIII:C activity increased by 30% in the vWf-deficient dogs, but there was only a slight change in vWf:Ag. Bleeding time decreased in 5 of 6 vWf-deficient dogs. In the normal dogs vWf:Ag increased by 14% after epinephrine infusion, but factor VIII:C activity did not change; neither parameter was altered in the vWf-deficient dogs. While the factor VIII:C/vWf:Ag complex was increased in the normal dog by exercise and DDAVP, the increase is not as pronounced as has been reported for humans. It is not known whether the poor response of the vWf-deficient dog is due to low levels of vWf in their endothelium or to a release defect.

Evaluation of canine red blood cells stored in a saline, adenine, and glucose solution for 35 days.

An additive solution for the storage of red blood cells was evaluated for use in dogs. Blood collected from 6 dogs was processed into packed red blood cells and stored for 35 days in the additive solution Nutricel (Miles, Inc, Pharmaceutical Division, West Haven, CT). Packed red blood cells stored in citrate-phosphate-dextrose-adenine (CPDA-1; Fenwal Laboratories, Baxter Health Care Corp, Deerfield, IL) also were evaluated for comparison. Red blood cell 2,3-diphosphoglycerate (2,3-DPG) concentration, adenosine triphosphate (ATP) concentration, percentage hemolysis, and pH were determined. The red blood cell post-transfusion viability (PTV) after 35 days of storage was assessed with both single-labeled chromium 51 (51Cr) and double-labeled technetium 99m/chromium 51 (99mTc/51Cr) techniques. Mean ATP concentration and percentage hemolysis of the cells stored in Nutricel were 1.1 mumol/g hemoglobin (Hb) and 0.28% respectively and did not differ significantly (P < .05) from the values of 1.0 mumol/g Hb and 0.33% from the CPDA.1-stored red blood cells. The mean pH of red blood cells stored in Nutricel was 6.19, which was significantly lower than the pH of 6.47 for cells stored in CPDA-1. The mean 2,3-DPG concentration of red blood cells stored in Nutricel was significantly higher at 10.1 mumol/g Hb than the 2,3-DPG concentration of 3.4 mumol/g Hb for cells stored in CPDA-1. The mean PTV of canine red blood cells stored in Nutricel for 35 days was 85% with 51Cr and 90% with 99mTc/51Cr. This was significantly higher than the mean PTVs of 38% and 36% for the CPDA-1 stored cells as assessed with 51Cr and 99mTc/51Cr techniques, respectively. It was concluded that 35-day-old canine red blood cells stored in Nutricel are of acceptable quality for transfusion purposes.

Use of a prestorage leukoreduction filter effectively removes leukocytes from canine whole blood while preserving red blood cell viability.

Leukoreduction of blood products is a technique used to prevent leukocyte-induced transfusion reactions. Filters currently used for human blood products achieve at least a 99.9% reduction in leukocyte numbers per unit (450 mL) of blood. Goals of this study were to determine if a prestorage leukoreduction filter could effectively achieve leukoreduction of canine blood and to determine if viability of the leukoreduced red blood cell (RBC) product could be maintained after 35 days of storage. Blood collected from each dog was filtered through a leukoreduction filter at either room temperature or after cooling (4 degrees C) for 4 hours. Filtration efficacy was determined by measurement of pre- and postfiltration leukocyte counts. In vitro viability of RBCs was determined by comparing RBC adenosine triphosphate concentration and percent hemolysis before and after the storage period. In vivo viability of stored cells was determined using a biotin-streptavidin-phycoerythrin labeling technique and flow cytometry. Blood filtered within 30 minutes of collection versus blood filtered after cooling had mean reductions in leukocyte numbers of 88.90 and 99.99%, respectively. The mean ATP and hemoglobin concentrations from the in vitro analysis were comparable to those obtained in previously for canine RBC adequately stored for 35 days. The mean in vivo 24-hour survival of the stored RBC was 84.7%. The leukoreduction filter used did not adversely affect in vitro or in vivo viability of canine RBCs. The filter effectively removed leukocytes from blood, with maximal efficiency of filtration achieved with use of cooled blood.

Evaluation of an additive solution for preservation of canine red blood cells.

The effect of an additive preservative solution on canine red blood cell posttransfusion viability (PTV) and on selected canine red blood cell biochemical parameters was studied. One unit (450 mL) of blood was collected from 6 clinically normal dogs into the anticoagulant citrate phosphate dextrose, centrifuged, and the plasma removed. The red blood cells were then suspended in 100 mL of a saline, adenine, dextrose, and mannitol solution and stored at 4 degrees C. Aliquots were removed for study at 1, 10, 20, 30, 37, and 44 days. The 24-hour PTV of autologous red blood cells was determined using a sodium chromate (51Cr) label. Red blood cell concentrations of 2,3-diphosphoglycerate (2,3-DPG), adenosine-5'-triphosphate (ATP), and pH were also determined. Canine red blood cell PTV, pH, ATP, and 2,3-DPG concentrations decreased during storage (P < .05). The PTV decreased from 94% using day 1 red blood cells to 80% and 75% using day 37 and day 44 red blood cells, respectively (P < .05). Although the mean PTV of the day 44 stored units equaled the Food and Drug Administration (FDA) minimum standard for human red blood cells, the PTV was substandard in 75% of the day 44 units. The FDA standard was exceeded in 83% of the day 37 units. It was concluded that 37-day-old canine red blood cells preserved with a saline, adenine, dextrose, and mannitol solution are of acceptable quality for transfusion.

Culture and characterization of equine terminal arch endothelial cells and hoof keratinocytes.

OBJECTIVES: To develop methods to isolate, culture, and characterize equine hoof endothelial cells (EC) and keratinocytes. SAMPLE POPULATION: Cells harvested from the forelimbs of 8 horses. PROCEDURE: EC were obtained via catheters placed in the palmar digital arteries of the disarticulated lower portion of the forelimbs from 4 horses that had been heparinized prior to euthanasia. Phosphate-buffered saline solution was used to remove and discard RBC from blood vessels, and collagenase was used to loosen and flush EC from the vasculature. Hoof keratinocytes were obtained from 4 recently euthanatized horses by use of dispase/trypsin dissociation of the coronary band epidermis. Use of an extracellular matrix gel as a culture flask attachment factor was important to the success of hoof keratinocyte cultures. RESULTS: EC from the palmar digital arteries were successfully cultured and characterized by in vitro morphology, uptake of a fluorescence-labeled acetylated-low density lipoprotein, and lack of expression of von Willebrand factor and smooth muscle actin. Hoof keratinocytes were characterized by morphology in culture and expression of keratin proteins, as determined by immunochemical reaction. Keratinocyte cultures were also positive for vimentin expression. CONCLUSIONS: Culture techniques to isolate and characterize hoof cells should aid investigators in their study of equine hoof pathobiologic features, especially as it relates to laminitis.

Use of an in vitro biotinylation technique for determination of posttransfusion viability of stored canine packed red blood cells.

OBJECTIVE: To determine posttransfusion viability (PTV) of canine RBC stored for 35 days in an additive solution, using in vitro biotinylation and technetium-99m and chromium-51 (99mTc/51Cr) labeling techniques. SAMPLE POPULATION: 6 random source, adult dogs. PROCEDURE: RBC from dogs were labeled with N-hydroxysuccinimide biotin (NHS-biotin) or 99mTc/51Cr in a crossover design. One unit (450 ml) of whole blood was collected from each dog, processed into packed RBC, and stored for 35 days in an additive solution. The process was repeated at a later date, so that each dog had 2 units stored under similar conditions. Stored autologous RBC were then labeled with either NHS-biotin or 51Cr and reinfused. When 51Cr was used, labeled cells were infused simultaneously with freshly drawn cells labeled with 99mTc. Posttransfusion viability of labeled cells was determined by dividing counts per minute (99mTc/51Cr) or percentage of cells (NHS-biotin) labeled at 24 hours by counts per minute or percentage of cells labeled after infusion. RESULTS: Mean PTV of packed RBC stored for 35 days in an additive system was 80% when determined by biotinylation, 83% as determined by 99mTc/ 51Cr, and 81% as determined by 51Cr alone. CONCLUSIONS: In vitro biotinylation provides an acceptable, nonradioisotopic means of determining PTV of stored canine packed RBC. CLINICAL RELEVANCE: NHS-biotin can be used to determine maximal storage time of canine RBC prepared for transfusion purposes.

Effects of intravenous administration of formaldehyde on platelet and coagulation variables in healthy horses.

OBJECTIVES: To assess safety and determine effects of IV administration of formaldehyde on hemostatic variables in healthy horses. ANIMALS: 7 healthy adult horses. PROCEDURE: Clinical signs and results of CBC, serum biochemical analyses, and coagulation testing including template bleeding time (TBT) and activated clotting time (ACT) were compared in horses given a dose of 0.37% formaldehyde or lactated Ringer's solution (LRS), IV, in a 2-way crossover design. In a subsequent experiment, horses received an infusion of 0.74% formaldehyde or LRS. In another experiment, horses were treated with aspirin to impair platelet responses prior to infusion of formaldehyde or LRS. RESULTS: Significant differences were not detected in any variable measured between horses when given formaldehyde or any other treatment. Infusion of higher doses of formaldehyde resulted in adverse effects including muscle fasciculations, tachycardia, tachypnea, serous ocular and nasal discharge, agitation, and restlessness. CONCLUSIONS AND CLINICAL RELEVANCE: Intravenous infusion of formaldehyde at doses that do not induce adverse reactions did not have a detectable effect on measured hemostatic variables in healthy horses.

Effects of sodium citrate, low molecular weight heparin, and prostaglandin E1 on aggregation, fibrinogen binding, and enumeration of equine platelets.

OBJECTIVE: To investigate the effects of sodium citrate, low molecular weight heparin (LMWH), and prostaglandin E1 (PGE1) on aggregation, fibrinogen binding, and enumeration of equine platelets. SAMPLE POPULATION: Blood samples obtained from 4 Thoroughbreds. PROCEDURE: Blood was collected into syringes in the ratio of 9 parts blood:1 part anticoagulant. Anticoagulants used were sodium citrate, LMWH, sodium citrate and LMWH, or 300 nM PGE1/ml of anticoagulant. Platelet aggregation in response to ADP, collagen, and PGE1 was assessed, using optical aggregometry. Platelet activation was evaluated, using flow cytometry, to detect binding of fluorescein-conjugated anti-human fibrinogen antibody. Plasma concentration of ionized calcium was measured, using an ion-selective electrode. RESULTS: Number of platelets (mean +/- SEM) in samples containing LMWH (109.5+/-11.3 x 10(3) cells/microl) was significantly less than the number in samples containing sodium citrate (187.3+/-30.3 x 10(3) cells/microl). Increasing concentrations of sodium citrate resulted in reductions in platelet aggregation and plasma concentration of ionized calcium. Addition of PGE1 prior to addition of an agonist inhibited platelet aggregation in a concentration-dependent manner, whereas addition of PGE1 4 minutes after addition of ADP resulted in partial reversal of aggregation and fibrinogen binding. CONCLUSIONS AND CLINICAL RELEVANCE: A high concentration of sodium citrate in blood samples decreases plasma concentration of ionized calcium, resulting in reduced platelet aggregation and fibrinogen binding. Platelets tend to clump in samples collected into LMWH, precluding its use as an anticoagulant. Platelet aggregation and fibrinogen binding can be reversed by PGE1, which may result in underestimation of platelet activation.

Effect of cryoprecipitate and plasma on plasma von Willebrand factor multimeters and bleeding time in Doberman Pinschers with type-I von Willebrand's disease.

We determined whether administration of cryoprecipitate or fresh-frozen plasma (FFP) would enhance glass bead platelet retention and shorten the bleeding time in von Willebrand factor (vWf)-deficient Doberman Pinschers. Plasma concentration of vWf was < 15% of the reference value in these dogs and, on the basis of multimeric analysis of vWf, these dogs had type-I von Willebrand's disease (vWd). Concentration of vWf in cryoprecipitate (prepared from FFP of clinically normal dogs) was enriched almost 20 times, and the preparation was a concentrate of the largest and most physiologically active multimers. Administration of a dose of cryoprecipitate calculated to increase plasma vWf concentration of recipient dogs to 50 U/dl increased plasma vWf concentration in recipient dogs to about 40 U/dl. Mean buccal mucosal bleeding time (BMBT) shortened from 6.7 minutes before treatment to 3.8 minutes at 2 hours after treatment. Cryoprecipitate from donor dogs treated with deamino-8-D-arginine vasopressin (1 micrograms/kg of body weight) effectively shortened mean BMBT from 6.4 minutes to 3.1 minutes. Administration of cryoprecipitate from vWf-deficient dogs prolonged, rather than shortened, the BMBT. After FFP (450 ml) infusion, plasma vWf concentration increased in recipient dogs, but the BMBT did not shorten. Glass bead platelet retention did not change after administration of cryoprecipitate or FFP.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative studies of erythropoiesis in the clinically normal, phlebotomized, and feline leukemia virus-infected cat.

Erythropoiesis was evaluated in 5 cats at base line with normal PCV and then in the same cats with anemia induced by phlebotomy and in 5 other cats with nonregenerative anemia from community-acquired feline leukemia virus (FeLV) infection. The hematologic evaluation included complete blood cell and reticulocyte counts, marrow morphologic features, determination of serum erythropoietin concentrations by radioimmunoassay, ferrokinetic studies, and in vitro marrow culture of early erythroid progenitors (erythroid burst-forming units; BFU-E) and late erythroid progenitors (erythroid colony-forming units; CFU-E). Phlebotomized cats developed marrow erythroid hyperplasia and an increased reticulocyte count. Ferrokinetic studies revealed an increase in plasma iron turnover from 1.4 to 3.8 mg of Fe/dl of blood/day and RBC use from 50.4% to 78.5%. The mean CFU-E number and CFU-E/BFU-E ratio increased after phlebotomy, but the increase was not significant (P greater than 0.05). Serum erythropoietin values did increase significantly. In FeLV-infected cats, a nonregenerative anemia was demonstrated by marrow erythroid hypoplasia and a low total reticulocyte count. An increased percentage of rubriblasts and prorubricytes was observed in 4 of the 5 cats. Although serum erythropoietin values were high (321 +/- 123 mU/ml vs normal 14 +/- 1 mU/ml), ferrokinetic data revealed decreased erythropoiesis. Marrow culture studies in the FeLV-infected cats also revealed low numbers of BFU-E and CFU-E, but normal numbers of granulocyte-macrophage progenitors remained. Seemingly, the FeLV infection impaired the ability of feline marrow to respond physiologically to anemia.

Hyperkalemia associated with potassium chloride administration in a cat.

Addition of appropriate amounts of potassium chloride solution to fluid administered i.v. resulted in hyperkalemia in a cat. To evaluate whether incomplete mixing of potassium chloride in the fluid might have resulted in the observed hyperkalemia, 40 mEq (20 ml) of potassium chloride solution was injected into each of three 1-L vinyl bags of 5% dextrose in water, with or without attempting to mix the additive with the fluid in the bag. Measurement of potassium concentrations in the bags revealed that injecting potassium chloride solution into a bag of fluid while that fluid is being administered can result in incomplete mixing and discharge of concentrated potassium chloride from the administration set. The greatest potassium concentration measured in fluid sampled from the administration set was 194 mEq/L.

Survival of pigeon red blood cells after transfusion into selected raptors.

Survival time of 51Cr-labeled pigeon RBC transfused into 5 raptors was determined. Mean +/- SD estimated RBC survival time was 0.51 +/- 0.19 days. This was considerably shorter than estimated survival time of autologous RBC in a Red-tailed Hawk (estimated survival, 35.1 days) and in a pigeon (estimated survival, 26.8 days). Estimated survival time after homologous transfusion of RBC from one pigeon to another was 7.1 days. Although single heterologous blood transfusions have been recommended as a safe and efficacious means of whole blood replacement in birds, results of this study suggest that heterologous RBC transfused from pigeons to selected raptor species are rapidly destroyed.

Prevalence and sequelae of feline leukemia virus infection in laboratory cats.

Over a 4-year period, 1,683 pound-source cats received at a research institution were screened for feline leukemia virus (FeLV) infection, using an indirect fluorescent antibody test. Viremia was detected in 83 of the cats, for a prevalence of 4.9%. During this period, FeLV infection was detected in 5 kittens on a research project; lymphoma or anemia developed 6 to 17 months after the infections were detected. It was concluded that apparently healthy cats infected with FeLV may not be appropriate for some biomedical research projects.

Selection of anticoagulant-preservatives for canine and feline blood storage.

Blood or blood component transfusions have become a well recognized, lifesaving form of therapy in veterinary medicine. Blood used for small animal transfusions may be collected and prepared with a variety of anticoagulants, anticoagulant-preservatives, or additive solutions. Selection of the most appropriate of these collection or storage solutions requires a knowledge of their formulations and of the shelf-lives previously established for storage of canine or feline red blood cells. Other factors that should be considered in the selection process are based on the specific transfusion needs of a clinic and its patients, including whether the blood will be used fresh or stored, the length of storage time desired, and whether components will be prepared. New products and techniques for blood storage continue to be developed, offering exciting new possibilities for the future practice of veterinary transfusion medicine.

Principles of transfusion medicine in small animals.

The purpose of this review was to provide the reader with an updated overview of small animal transfusion medicine, and an approach to integrating it into private practice, based on a review of the veterinary and human literature spanning the last 3 decades. Electronic, online databases that were searched included CAB International and Medline; multiple keywords or subject headings were searched that were appropriate to each of the sections reviewed: canine and feline blood groups, blood-typing and crossmatching, donors, blood collection, storage, blood components, blood transfusion, blood component therapy, blood substitutes, and adverse reactions. The safe use of blood component therapy requires knowledge of blood groups and antibody prevalence, and knowledge of the means to minimize the risk of adverse reactions by including the use of proper donors and screening assays that facilitate detection of serological incompatibility. The 2 assays available to the practitioner are crossmatching, which is readily done in-house, and blood typing. Blood typing is available in the form of a commercial testing kit, through use of purchased reagents, or via a request to an external laboratory. The risk of potentially fatal adverse reactions is higher in cats than in dogs. The decision to transfuse and the type of product to administer depend on several factors, such as the type of anemia and the size of the animal. In conclusion, transfusion medicine has become more feasible in small animal practice, with improved access to blood products through either on-site donors, the purchase of blood bank products, external donor programs, or the availability of blood component substitutes.

Changes in arterial, mixed venous and intraerythrocytic ion concentrations during prolonged exercise.

REASONS FOR PERFORMING STUDY: Prolonged equine exercise can cause hypochloraemic alkalosis and hypokalaemia secondary to the loss of hypertonic sweat. Movement of ions in and out of erythrocytes during exercise may help regulate acid-base balance and changes in plasma ion concentrations. The extent to which this happens during prolonged equine exercise has not been reported. OBJECTIVES: To measure changes in blood gases and major plasma and intraerythrocytic (iRBC) ion concentrations of horses undergoing prolonged submaximal exercise. METHODS: Six horses were trotted at ∼ 30% VO2max on a treadmill for 105 min. Arterial ((a)) and mixed venous ((v)) blood samples were collected every 15 min, and pre- and post exercise. Blood gases and plasma (pl) concentrations of sodium, potassium, chloride and protein were measured and their iRBC concentrations calculated and compared (P < 0.05). RESULTS: P(a)CO(2) decreased in all horses. pl[Cl(-)]v decreased and [HCO(3)(-)]v increased. Due to the exhalation of CO(2) and chloride shifting, [HCO(3)(-)]a<[HCO(3)(-)]v, pl[Cl(-)]a>pl[Cl(-)]v)and iRBC[Cl(-)]aiRBC[K(+)]v. Conversely, iRBC[Na(+)]a