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 μmol/g hemoglobin (Hb) and 0.28% respectively and did not differ significantly (P < .05) from the values of 1.0 μmol/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 μmol/g Hb than the 2,3-DPG concentration of 3.4μmol/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.     

Comparison of 4 blood storage methods in a protocol for equine pre-operative autologous donation

OBJECTIVE: To compare viability of equine whole blood stored by 4 different methods, and to establish optimal storage protocols for an equine autologous blood donation program. STUDY DESIGN: In vitro study of stored equine whole blood. Animals- Six healthy adult horses. METHODS: Blood from each horse was collected into 4 different containers: glass bottles containing acid-citrate-dextrose solution (ACD), plastic bags containing ACD, citrate-phosphate-dextrose (CPD), and CPD with supplemental adenine (CPDA-1). Blood was stored for 5 weeks and sampled at 2-day intervals. Standard hematologic and biochemical variables were evaluated, and adenosine-5-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) concentrations were measured and normalized to total hemoglobin content. RESULTS: Plasma hemoglobin, % hemolysis, lactate, potassium, ammonia, and lactate dehydrogenase (LDH) increased, whereas glucose concentration and pH decreased in all stored blood over 5 weeks. There was a temporal increase in hemolysis with all storage methods, but the increase was greatest in glass bottles. Lactate and ammonia were highest in CPD and CPDA-1 samples, indicating more active red blood cell (RBC) metabolism. 2,3-DPG concentrations decreased during storage, but were optimally preserved with CPDA-1. ATP concentrations were significantly higher for blood stored in CPDA-1, and were lowest in glass bottles. CONCLUSIONS: Hematologic and biochemical values measured for blood stored in CPDA-1 are suggestive of improved RBC viability compared with other storage methods. With the exception of ATP, results from stored equine blood were similar to those reported for other species. CLINICAL RELEVANCE: Commercial CPDA-1 bags appear to be the optimal storage method for equine whole blood.     

Storage of equine red blood cells as a concentrate

The study was undertaken to determine how equine red blood cells (RBCs) survive in storage bags designed for use with human RBCs. Separated RBCs were stored in a routine manner for 35 days and examined every 7 days for storage lesions. Measured parameters included haematology, haemolysis, pH, potassium, lactate, adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG). All tests were performed in vitro. Haematology did not change significantly. Haemolysis increased during storage but did not exceed human limits. pH and 2,3-DPG decreased, while lactate, potassium and ATP increased. RBCs deteriorated somewhat during storage, but when compared with human in vitro parameters, remained suitable for transfusion. It is concluded that equine erythrocytes can be stored for at least 35 days before transfusion.     

Evaluation of Citrate-Phosphate- Dextrose-Adenine as a Storage Medium for Packed Canine Erythrocytes

Packed canine red blood cells (RBCs) stored in the anticoagulant-preservative solution citrate-phosphate-dextrose-adenine (CPDA-1) were studied at 1, 10, 20, 30, and 40 days. The extracellular concentrations of potassium and sodium, erythrocyte mean corpuscular volume, and osmotic fragility increased during storage (P less than 0.05). There was a decrease in the pH, plasma concentration of glucose, and erythrocyte concentrations of 2,3-diphosphoglycerate (2,3-DPG) and adenosine-5'-triphosphate (P less than 0.05). Erythrocyte 2,3-DPG concentration decreased by 54% within the first 24 hours of storage (P less than 0.001). Posttransfusion viability (PTV) decreased from 90% on day 1 to 46% on day 40 (P less than 0.05). The PTV of the RBCs stored for 10 and 20 days complied with the Food and Drug Administration (FDA) standard. Although there are marked biochemical and hematologic changes in stored packed red blood cells (pRBCs), 20-day-old units may be expected to be of acceptable quality. The sharp decrease in 2,3-DPG concentration suggests a reduction in oxygen carrying capacity in erythrocytes stored as pRBCs. Hyperkalemia occurs during storage of pRBCs and does not appear to be associated with high intraerythrocytic potassium concentrations.     

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°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 (⁶¹Cr) 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.     

Hypophosphatemia

Severe hypophosphatemia (i.e., serum phosphorus concentration below 1 mg/dl) occurs infrequently in veterinary patients. It is most often associated with diabetic ketoacidosis in small animals. Phosphate is necessary for the production of 2,3 diphosphoglycerate (2,3-DPG) and adenosine triphosphate (ATP); both are important for normal cellular metabolism. Consequences of severe hypophosphatemia may include hemolytic anemia, seizures, altered mentation, cardiomyopathy, and skeletal muscle weakness. Parenteral phosphate therapy is necessary in most cases of severe hypophosphatemia.     

Bovine post-parturient haemoglobinuria: effect of inorganic phosphate on red cell metabolism

Washed red blood cells from normal cows were incubated as 10 and 20 per cent suspensions in media containing 0, 2.5 and 25 mM phosphate. The results showed that the rate of glycolysis was dependent on the inorganic phosphate concentration. In the absence of phosphate, the consumption of glucose and the production of 2,3-diphosphoglycerate, lactate and adenosine 5'-triphosphate (ATP) were decreased. Incubation without added phosphate also greatly increased the production of fructose-1,6-diphosphate, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Moderate hypophosphataemia was induced in two pre-ruminant calves. Washed red cells from the blood of these animals showed a depletion of ATP when compared with red cells from a control calf. The results indicate that phosphorus deficiency, leading to hypophosphataemia, may be a mechanism of post parturient and related syndromes of haemoglobinuria by decreasing red cell glycolysis and resultant ATP synthesis. Subnormal concentrations of ATP would predispose red cells to altered structure and function, a loss of normal deformability, and an increase in fragility and haemolysis with resultant haemoglobinuria.     

Oxygen affinity responses to 2,3-diphosphoglycerate, and methaemoglobin formation in horse and human haemoglobins.

The oxygen affinities of horse and human haemoglobins were compared in the absence and presence of the allosteric effector 2,3-diphosphoglycerate (2,3-DPG). Horse haemoglobin solutions showed significantly smaller responses to the presence of 2,3-DPG, and this difference may be due to different amino acid substitutions at position NA2(2)beta. Horse haemoglobin solutions from erythrocytes containing different ratios of the two different haemoglobin types showed similar oxygen affinities in the absence and presence of 2,3-DPG. Horse haemoglobins in solution were found to autoxidise to methaemoglobin much more readily than human haemoglobin under the same conditions, and this is an important consideration when measuring the oxygen affinity of horse haemoglobin solutions. This difference could be due to different amino acid residues at position NA2(2)beta.     

Pseudohyperkalemia in Akitas

Hyperkalemia was observed in a clinically normal Akita with normal ACTH stimulation test results, endogenous ACTH concentration, and SUN concentration. A diagnosis of pseudohyperkalemia caused by red cell hemolysis was made by comparing plasma potassium content from hemolyzed and non-hemolyzed specimens. Red cell potassium content was measured in 8 Akitas and 6 non-Akita control dogs. Six of 8 Akitas had high erythrocyte potassium content relative to their plasma and to that of the control dogs. Five of eight Akitas had microcytosis. Plasma from affected dogs had high potassium content after being refrigerated in contact with red cells for 4 hours or more. The increase in plasma potassium content was progressive with prolonged contact with the red cells and was accompanied by a decrease in plasma sodium content.     

Effect of storage on oxygen dissociation of canine blood.

Oxygen dissociation curve and adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG) contents in red blood cells (RBC) were determined in canine blood stored in acid citrate dextrose (ACD) and citrate phosphate dextrose (CPD) solutions. The oxygen-unloading ability decreased, as shown by the left shift of oxygen dissociation curve during storage, and the shift correlated with decreasing DPG but not decreasing ATP concentrations. After 2 weeks of storage in ACD solution, oxygen dissociation curves were shifted significantly to the left. For blood stored in CPD solution, 4 weeks was required before the shift was significant. It was concluded that canine blood collected and stored in CPD solution is more efficient than that stored in ACD solution in delivering oxygen to the tissues.     

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.     

Mechanisms controlling the oxygen consumption in experimentally induced hypochloremic alkalosis in calves

The study was carried out on healthy Friesian calves (n = 10) aged between 10 and 30 days. Hypochloremia and alkalosis were induced by intravenous administration of furosemide and isotonic sodium bicarbonate. The venous and arterial blood samples were collected repeatedly. 2,3-diphosphoglycerate (2,3-DPG), hemoglobin and plasmatic chloride concentrations were determined. The red blood cell chloride concentration was also calculated. pH, PCO2 and PO2 were measured in arterial and mixed venous blood. The oxygen equilibrium curve (OEC) was measured in standard conditions. The correspondence of the OEC to the arterial and mixed venous compartments was calculated, taking blood temperature, pH and PCO2 values into account. The oxygen exchange fraction (OEF%), corresponding to the degree of blood desaturation between the arterial and mixed venous compartments and the amount of oxygen released at the tissue level by 100 mL of blood (OEF Vol%) were calculated from the arterial and mixed venous OEC, combined with PO2 and hemoglobin concentration. Oxygen delivery (DO2) was calculated using the arterial oxygen content, the cardiac output measured by thermodilution, and the body weight of the animal. The oxygen consumption (VO2) was derived from the cardiac output, OEF Vol% and body weight values. Despite the plasma hypochloremia, the erythrocyte chloride concentration was not influenced by furosemide and sodium bicarbonate infusion. Due to the alkalosis-induced increase in the 2,3-DPG, the standard OEC was shifted to the right, allowing oxygen to dissociate from hemoglobin more rapidly. These changes opposed the increased affinity of hemoglobin for oxygen induced by alkalosis. Moreover, respiratory acidosis, hemoconcentration, and the slight decrease in the partial oxygen pressure in mixed venous blood (Pvo2) tended to improve the OEF Vol% and maintain the oxygen consumption in a physiological range while the cardiac output, and the oxygen delivery were significantly decreased. It may be concluded that, despite reduced oxygen delivery, oxygen consumption is maintained during experimentally induced hypochloremic alkalosis in healthy 10-30 day old calves.     

Comparison of Optisol-GS and neomycin-polymyxin B-gramicidin ophthalmic solution for corneal storage in the dog

The objective of the research was to compare the efficacy of Optisol-GS (OGS, Bausch & Lomb Surgical, Irvine, CA, USA) with triple antibiotic ophthalmic solution (neomycin-polymyxin B-gramicidin, NPG; Bausch & Lomb, Tampa, FL, USA) in preserving the viability of corneal endothelial cells. The study subjects were thirty young to middle-aged dogs with no gross corneal pathology that had been euthanized by pentobarbital overdose for reasons unrelated to this project. Corneal tissues were harvested, analyzed, and randomly assigned to treatment groups: one of two media (OGS or NPG), and one of five storage times (1, 7, 14, 21, or 35 days). Six corneas were stored in each medium for each time period. Corneal endothelial cell viability was evaluated pre- and poststorage by vital staining (trypan blue and alizarin red S), and endothelial cell morphology was evaluated with scanning electron microscopy. Storage in NPG caused significant loss (100%) of endothelial cells after all storage times. OGS storage maintained a high level of endothelial cell viability up to 21 days (98.9% ± 1.3% viability). A significant decrease in percentage viability was also found for OGS-stored corneas between 21 and 35 days, when endothelial cell viability decreased to 61.4% ± 45.9%. The conclusions are that NPG storage at −20 °C is a very poor choice of media for corneal tissue banking if graft clarity is the goal. Storage in Optisol-GS at 4 °C for up to 21 days resulted in significantly higher percentages of viable endothelial cells. Optisol-GS storage should facilitate corneal preservation for canine keratoplasty patients.     

Relationship between barnacle epibiotic load and hematologic parameters in loggerhead sea turtles (Caretta caretta), a comparison between migratory and residential animals in Pamlico Sound, North Carolina.

Health status of a total of 57 loggerhead sea turtles (Caretta caretta; 42 migratory and 15 residential turtles) was analyzed using body condition and hematologic parameters. A subset of 18 juvenile migratory loggerhead sea turtles in the fall of 1997 and 15 residential turtles in the summer of 2000 were analyzed for barnacle epibiota. The migratory group had significantly higher red blood cell counts and percent heterophils and significantly lower percent lymphocyte and absolute eosinophil counts, as well as significantly lower plasma concentrations of calcium, sodium, chloride, potassium, glucose, alkaline phosphatase, and anion gap. Many of these variations may be because of physiology of migration. A positive association between turtle weight and hematocrit was detected and may be because of larger turtles diving for longer periods of time. There were no significant differences of epibiota load, health of the turtles, or condition index between turtles captured during the two events.     

Some aspects of erythrocyte metabolism in a dog with polycythaemia vera

An 11-year-old female crossbred dog showed signs of polyuria, polydipsia, vomiting, posterior weakness and ataxia. Clinical and laboratory findings suggested the diagnosis of polycythaemia vera. The haematological values shown over a six-month period are presented. In four samples some aspects of erythrocyte function (glucose-6-phosphate dehydrogenase [G6PD] and pyruvate kinase [PK] activities, 2,3 diphosphoglycerate [2,3 DPG] concentration, osmotic fragility and intracellular sodium and potassium concentrations) were studied. Variable activities of G6PD and PK, probably related to different reticulocyte number, were detected together with normal osmotic fragility and intracellular sodium and potassium concentrations. 2,3 DPG concentration was higher than normal in all four samples. This could be interpreted as a response to a low tissue perfusion rather than a higher content of 2,3 DPG in red blood cells from the polycythaemic dog.     

Hypophosphatemia Induced by Dietary Aluminium Hydroxide Supplementation in Growing Pigs: Effects on Erythrocytes,Myocardium, Skeletal Muscle and Liver

Håglin, L., B. Essén-Gustavsson and A. Lindholm: Hypophosphatemia induced by dietary aluminium hydroxide supplementation in growing pigs: Effects on erythrocytes, myocardium, skeletal muscle and liver. Acta vet. scand. 1994, 35, 263-271.– Three groups of pigs were studied during and after 10 weeks of treatment with either Al(OH)₃ (Al[OH]₃-group, n=8) to induce hypophosphatemia, A1P0₄ (AlP0₄-group, n=8, aluminium control without hypophosphatemia) or no addition to the feed (control group, n=8). Blood samples were taken at the start of the experiment and after 3, 6 and 10 weeks and were analyzed for phosphate, calcium and 2,3-diphosphoglycerate (2,3-DPG). Samples from myocardium, skeletal muscle and liver were obtained in connection with exsanguination and analyzed for glycogen, adenosine-tri-phosphate (ATP), creatine phosphate (CP), glucose-6-phosphate (G-6-P) and lactate. The Al(OH)₃-group became hypophosphatemic and hypercalcémie with low levels of 2,3-DPG in erythrocytes within 3 weeks and showed a retarded growth rate. After 10 weeks the Al(OH)₃-group had low levels of ATP in myocardium as compared with the control-group and low levels of G-6-P as compared with the AlP0₄-group. No disturbances on electro-cardiograms registered at rest could be documented. G-6-P concentration was low in the biceps muscle in the Al(OH)₃-group as compared with the AlP0₄-group and in the liver low G-6-P concentration was seen in addition to high lactate concentration. The fibre type composition in M. Longissimus did not differ between groups, but the Al(OH)₃-group had, due to retardation in growth, smaller mean fibre-areas than pigs in the AlP0₄-group. Hypophosphatemia gave rise to high serum calcium levels, low concentration of 2,3-DPG in erythrocytes and influenced G-6-P concentration in skeletal muscle, G-6-P and ATP in myocardium, G-6-P and lactate in liver. Retarded growth was one serious consequence of hypophosphatemia and the disturbed energy metabolism.     

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.     

Comparative haematology and blood chemistry of endangered lizards (Gallotia species) in the Canary Islands

Blood samples were taken from the ventral coccygeal vein of 15 El Hierro giant lizards (Gallotia simonyi) (seven females and eight males), six La Gomera giant lizards (Gallotia bravoana) (four males and two females) and four Tenerife giant lizards (Gallotia intermedia) (two males and two females), and 31 blood parameters were measured. Among the haematological parameters there were significant differences between the three species in heterophils, azurophils and lymphocytes, but no significant differences in red blood cell count, white blood cell count, haemoglobin, packed-cell volume, monocytes, eosinophils and basophils. In terms of blood chemistry there were significant differences between the three species in cholesterol, triglycerides, glucose, sodium, chloride, urea, uric acid, total proteins, prealbumin, albumin and gamma globulins, but no significant differences in calcium, potassium, aspartate aminotransferase, alanine aminotransferase, creatine kinase, bile acids, alpha-1 and alpha-2 globulins and beta globulins.     

Effects of standard anticoagulants and storage procedures on plasma glucose values in seals.

Standard methods for the preservation of vertebrate blood samples for glucose analysis include collecting and storing the blood in evacuated tubes containing sodium fluoride (glycolytic inhibitor) and potassium oxalate (anticoagulant). We found that blood collected from 5 seals by venipuncture and transferred into these tubes had a significantly (P less than 0.05) lower plasma glucose value than blood transferred into tubes containing heparin. In species in which RBC glucose concentration is significantly less than that in the plasma, fluoride and oxalate-induced hemolysis dilutes the plasma with cytoplasm and lowers the measured concentration of glucose in plasma. Therefore, although plasma glucose is used extensively in experimental and clinical analyses, standard techniques for handling the blood may create errors that could confuse comparisons between individuals or species.     

Evaluation of platelet additive solution for prolonging storage of functional canine platelet concentrate

Objective

To assess storage lesion development, platelet function, and bacterial growth in canine platelet concentrates (PCs) stored in a platelet additive solution (PAS) or a plasma control at 4°C for 21 days.

Design

Prospective, ex vivo, experimental controlled study.

Setting

University veterinary teaching hospital.

Animals

Ten units of canine PCs collected from blood bank donations.

Interventions

The PCs were separated into 2 bags, 1 containing 100% plasma and the other containing 35% plasma and 65% of a PAS (Plasma-Lyte A), and stored at 4°C for 21 days. At days 0, 7, 14, and 21, PCs were analyzed for the presence of swirling, aggregate formation, platelet counts, platelet indices, glucose, lactate, lactate dehydrogenase, Pvco ₂, Pvo ₂, aggregation via light aggregometry, activation percentages using flow cytometry, and bacterial growth.

Measurements and main results

Cold-stored PCs in both PAS and plasma control maintained mean pH >6.8 and mean lactate <9.0 mmol/L over 21 days, with no difference in glucose utilization. Swirl was maintained in both solutions for most days (76/80 combined total samples), with no difference in aggregate formation between solutions. The Pvco ₂ was higher in plasma on all days (P < 0.001), with no difference in Pvo ₂. Platelet indices did not reflect significant storage lesion development in either solution. Lactate dehydrogenase did not differ between solutions but did increase from day 7 to day 21. Mean maximal aggregation percentage was reduced overall but with no significant difference between solutions. The only observed difference in mean activation percentage between solutions was in PAS on day 7, which was significantly higher than plasma (P < 0.05). No bacterial growth occurred during storage.

Conclusions

Cold storage in PAS and plasma allowed PCs to be stored for up to 21 days with minimal storage lesion development, maintenance of platelet function, limited platelet activation, and no bacterial growth within stored bags.