Selection of Anticoagulant-Preservatives for Canine and Feline Blood Storage

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SELECTION OF ANTICOAGULANTPRESERVATIVES FOR CANINE AND FELINE BLOOD STORAGE K. Jane Wardrop, DVM, MS

Blood stored for transfusion purposes is typically collected into solutions that provide both anticoagulation and preservation of red blood cells. Transfused red blood cells must be able to survive in the circulation of the recipient and carry oxygen effectively to the tissues. The ideal anticoagulant-preservative would allow red blood cells to be stored for an unlimited length of time without impairment of viability or function. This ideal is unlikely to be achieved; however, careful selection of anticoagulant-preservatives can increase the length of time that blood or blood products can be stored and can lessen the adverse effect of storage on the red blood cell. HISTORICAL PERSPECTIVES

In the early days of human transfusion medicine, anticoagulants were unavailable and blood had to be removed quickly from the donor and administered to the recipient before clotting occurred. The procedure of choice for transfusions was thus direct transfusion via arteriovenous anastomosis. The development of sodium citrate and use of equal volumes of a sodium citrate and dextrose solution and blood during World War I completely transformed the practice of transfusion medi-

From the Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, Washington

VETERINARY CLINICS OF NORTH AMERICA: SMALL ANIMAL PRACTICE VOLUME 25 • NUMBER 6 • NOVEMBER 1995

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cine, allowing whole blood to be stored for human use for up to 21 days. 36 Further modifications of the citrate solution occurred during World War II, when Loutit and Mollison developed acid-citrate-dextrose (ACD) solution. 25 Citrate-phosphate-dextrose (CPO) was introduced in 1957, and differed from ACD in its slightly higher pH. In the early 1960s plastic bag systems were developed for blood storage, paving the way for component therapy. The anticoagulant-preservative citrate-phosphate-dextrose-adenine (CPDA-1), which utilizes extra adenine and dextrose, was approved for 35-day storage of human packed red blood cells by the Food and Drug Administration (FDA) in August 1978. Additive solutions were developed in the 1980s, and are currently FDA approved for 42 days of red blood cell storage. The development of these anticoagulant-preservatives for human transfusions also hastened their use in veterinary medicine. During the 1950s, glass vacuum bottles containing ACD were used for animal blood collection and subsequent transfusion. The availability of CPO in plastic bags later offered an alternative to the use of ACD and became a popular choice for use in animal whole blood transfusions. Plastic bag systems containing CPDA-1 were used in the 1980s and are still used by those veterinary institutions and practices utilizing component therapy. Today, additive solutions in plastic bag systems are available to the veterinary profession and are becoming increasingly popular for use in canine component therapy. RED BLOOD CELL METABOLISM AND THE RED BLOOD CELL STORAGE LESION

Although blood or its components may be used fresh, storing or banking the blood is advantageous for optimum retrieval. All red blood cell preservatives designed for the storage or banking of blood are formulated to minimize the effect of storage on the delicate biochemical balance of the red blood cell. Particular attention has been given to the ability of these preservatives to maintain red blood cell adenosine triphosphate (ATP) concentrations, and to a lesser extent, 2,3-diphosphoglycerate (2,3-DPG) concentrations. Red blood cells require energy in the form of ATP to maintain the normal shape and deformability of the red blood cell, to maintain appropriate intracellular cation concentrations, and to participate in phosphorylations and other reactions necessary for maintaining red blood cell membrane phospholipids. This energy is derived almost entirely from the breakdown of glucose to lactate through the glycolytic or Embden-Meyerhof pathway (Fig. 1). In this pathway, glucose is first phosphorylated by hexokinase, then proceeds through a series of reactions that divide the six-carbon sugar into two three-carbon sugars. The three-carbon sugars are again phosphorylated. Energy is required in steps involving the six-carbon carbohydrates and is generated in steps involving the three-carbon carbohydrates so that two ATPs are generated for every glucose that enters the pathway. Post-

SELECTION OF ANTICOAGULANT-PRESERVATIVES FOR BLOOD STORAGE

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Embden-Meyerhof Pathway Glucose

Hexose Monophosphate Shunt

ATP~ 1Hexokmase .

ADP

NADP+

NADPH

Glucose 6-P-----+--V.;;..________ 6-P-Giuconate

t

Glucose-6-phosphate Isomerase

Fructose 6-P ATP ' A~~

t

Aldolase

A,...,.... ..........

.........................

1(

NADP+ NADPH

..........

,....,....

_... _,-"

,. . ,. . ,. .

~c::

,. -' ...... phosphate isomerase

Glyceraldehyde-3-P

t

6-Phosphogluconate dehydrogenase

----------------------;:;:::'~Ribulose-5-P

1Phosphofructokinase .

Fructose 1,6-diP

NAD+ "'""\ NADH ~

Glucose-6-phosphate dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase

Dihydroxyacetone-P

Lueberi ng-Rapaport Pathway

1,3-diP-Giycerate - - - + - - - - - - - - - - - - - .1 Diphosphoglycerate mutase , ADP) Phosphoglycerate kinase 2 3-diP-Giycerate ATP

t

'

3-P-Giycerate

t

I

Diphosphoglycerate phosphatase

Phosphoglycerate mutase

2-P-Giycerate

t

Enolase

P-Enolpyruvate

ADP~ 1Pyruvate kinase ATP Pyruvate NADH)l Lactate dehydrogenase NAD+

Lactate Figure 1. Pathways of red blood cell metabolism.

storage ATP concentrations have been used to predict in vivo red blood cell viability, that is, the ability of the cell to circulate normally in a recipient after transfusion. The fall in ATP concentrations in the red blood cell during storage is of particular significance in view of studies involving the addition of various purine nucleosides and adenine to stored blood. 10• 30• 40 When adenine is added at the beginning of the storage period, both the decline in ATP concentrations and in post-transfusion red blood cell survival are diminished. The Embden-

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Meyerhof pathway also generates NADH, which is used to maintain hemoglobin in its reduced functional state. Some glucose metabolism by red blood cells occurs in the pentose phosphate/hexose monophosphate pathway (see Fig. 1). Some of the glucose 6-phosphate formed when glucose is phosphorylated in the hexokinase reaction may enter this pathway. This pathway is of importance to the red blood cell primarily as a source of NADPH, an essential compound in protecting the red blood cell against oxidative damage. 6 The production of large quantities of 2,3-DPG is a unique feature of glycolysis in the red blood cell. 2,3-DPG is produced by a special side pathway that branches from the main glycolytic pathway after the formation of 1,3-diphosphoglycerate and returns to it with the formation of 3-phosphoglyceric acid (see Fig. 1). 2,3-DPG binds to the beta subunits of deoxyhemoglobin, stabilizing the low-affinity conformation. 4 Elevation of 2,3-DPG decreases the oxygen affinity, resulting in the release of more oxygen at a given pH and p02 • 2,3-DPG is depleted early in blood storage, and preservatives that maintain 2,3-DPG at higher levels are desirable. Feline red blood cells have very low 2,3-DPG concentrations; 2,3-DPG is not required for the release of oxygen from feline hemoglobin.5 Cellular changes, such as the above described ATP and 2,3-DPG depletions, occur during red blood cell storage at 1oc to 6°C. These changes affect cell viability and function and ultimately contribute to the cells' diminished survival after transfusion. The changes are known collectively as the "storage lesion" of blood. Physical changes include shape alterations and vesiculation of the red blood cell membrane. 15' 16' 22 Changes in the protein structure of the red blood cell membrane can occur. 32, 38 Several quantitative changes in red blood cell biochemistry have also been noted after storage. Plasma glucose decreases as it continues to be metabolized during the storage period. The accumulation of lactic and pyruvic acids from glycolysis results in a decrease in the pH of the stored cells. This slows the rate of glycolysis, reducing both the red blood cell ATP and 2,3-DPG concentrations. The oxygen dissociation curve shifts to the left as the 2,3-DPG concentration decreases. 13 Much of the storage lesion is reversible after transfusion; however, the changes in membrane structure and deformability are irreversible. z, 45 Identifying even the reversible changes is important as their correction may impose excessive metabolic demands on an already compromised patient. EVALUATION OF ANTICOAGULANT-PRESERVATIVE SOLUTIONS

Extensive work has been done with human blood to develop storage media that retard development of the storage lesion and effectively extend the storage time of red blood cells. To be useful to the recipient, stored red blood cells that are transfused must survive in the circulation and carry oxygen efficiently to the tissues. Evaluation of cell viability in

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SELECTION OF ANTICOAGULANT-PRESERVATIVES FOR BLOOD STORAGE

a given storage medium is performed by both in vitro and in vivo studies. Commonly used in vitro indicators of red blood cell survival after storage include red blood cell ATP and 2,3-DPG concentrations, pH, and percent hemolysis. Supernatant sodium, potassium, and glucose concentrations can also be determined (Table 1). In vitro testing, however, cannot accurately predict how a given sample of stored red blood cells will survive in a recipient. This can be determined only by in vivo evaluation, where the stored cells are labeled and followed in the circulation. When red blood cells that have been stored are transfused, some cells are cleared within a few hours but the rest survive normally. 44 Knowledge of the percentage survival at 24 hours makes possible a prediction of whole population survival. The maximum allowable storage time for red blood cells in a given storage media, also termed the "shelf-life", is currently determined by in vivo post-transfusion viability studies. Current FDA requirements for stored human whole blood or red blood cells state that at least 75% of the transfused cells must remain viable 24 hours after transfusion if a preservative is to be considered satisfactory. 47 The in vivo evaluation of viability of stored red blood cells is accomplished by radiolabeling the cells, generally with 51-chromium ( 51 Cr), infusing them into a recipient, and following their fate over time in the circulation. 29 Post-transfusion recovery is then determined by expressing the 51 Cr activity measured in the circulation 24 hours after infusion as a percentage of that estimated to be present at the time of initial transfusion, based on the quantity of radionuclide injected and the recipient's total red blood cell mass. The simplest way to perform a measurement of red blood cell mass is by "back-extrapolation" of the Table 1. HEMATOLOGIC AND BIOCHEMICAL PARAMETERS OF CANINE PACKED RED BLOOD CELLS STORED IN CPDA-1 AND ADSOL*

CPDA-1 Parameter Packed cell volume (%) Plasma hemoglobin (mg/dL) Total hemoglobin (g/dL) Hemolysis (%) ATP (J.Lmol/g Hb) 2,3-DPG (J.CmOI/g Hb) pH Glucose (mg/dL) Sodium (mEq/L) Potassium (rnEq/L)

Day 0 73 30 24.3 0.03 1.69 17.19 6.98 656 188 3.3

± ± ± ± ± ± ± ± ± ±

2

Adsol Day 35

72 ± 9 283 ± 202 10 22.2 22.2 ± 3.2 0.01 0.33 ± 0.23 0.37 0.92 ± 0.20 3.09 4.47 ± 3.19 0.04 6.47 ± 0.05 42 76 ± 87 5 215 ± 17 8.6 ± 1.9 0.2

Day 0 60 ± 22 ± 23.2 ± 0.03 ± 1.72 ± 16.87 ± 7.02 ± 1370 ± 147 ±

2 9

2.0 0.01 0.24t 2.08 0.02 132 5 1.0 ± 0.1

Day 35 58 139 21.8 0.26 1.20 7.56 6.26 715 171 5.6

± 8 ± 49 ± 4.4

± ± ± ± ± ± ±

0.07 0.25 1.79 0.13 76 6 0.5

CPDA-1 = Citrate-phosphate-dextrose-adenine; Hb = hemoglobin. *n = 6; all values expressed as mean ± SD. tn = 5. Adapted from Wardrop KJ, Young J, Wilson E: An in vitro evaluation of storage media for the preservation of canine packed red blood cells. Veterinary Clinical Pathology 23:84-86, 1994; with permission.

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survival curve to a time zero, using the single-isotope (51Cr) technique. Blood samples are drawn at frequent intervals after the infusion of 51Crlabeled stored blood. The logarithm of the radioactivity of the samples is plotted against time and back-extrapolated to time zero. The intercept at time zero is assumed to represent 100% survival of cells. This backextrapolation must be based on the assumption that the exponential rate of loss during the sampling period occurs at a constant rate. A rapid early loss of infused cells thus results in a falsely high estimate of the red blood cell mass and a spuriously high red blood cell viability estimate at 24 hours. This problem can be overcome by making an independent measurement of the red blood cell mass using a different isotope, such as 99m-technetium (99mTc). Freshly drawn cells labeled with 99 mTc and stored cells labeled with 51 Cr are simultaneously infused into the recipient, and the red blood cell viability based on a 99mTcdetermined red blood cell mass is calculated. 18 Nonradioisotopic labels such as biotin also show promise for use in viability studies, and may provide a safe and effective alternative to radioisotopes in the future. 6 • 8• 44 ANTICOAGULANT-PRESERVATIVES USED IN SMALL ANIMAL TRANSFUSIONS

The anticoagulants, anticoagulant-preservatives, and additive solutions described in the following text are available for use in small animal transfusions. Veterinary blood banks, such as the Animal Blood Bank (Dixon, CA) and Hemopet (Irvine, CA) carry a number of these products as well as infusion supplies. Major suppliers of single and multiple blood pack systems for human transfusion needs include the Baxter Healthcare Corporation, Deerfield, IL; Miles, Pharmaceutical Division, West Haven, CT; and the Terumo Medical Corporation, Somerset, NJ. Though the majority of anticoagulant-preservatives and additive solutions were originally designed for human use, they can be quite satisfactory for use in veterinary transfusions. However, the shelf-life of red blood cells in a given storage medium differs among species, and the shelf-lives indicated for human cells must not be extended to canine or feline blood cells. Anticoagulants

Heparin has been used for the transfusion of fresh whole blood to cats and small dogs. Heparin prevents coagulation by combining with endogenous antithrombin III (ATIII). The ATIII-heparin complex is a potent inhibitor of serine proteases involved in the coagulation cascade, including thrombin and factors IXa, Xa, Xla, and Xlla. 9 Heparin has no preservative properties and heparinized blood should be transfused shortly after collection. Generally a dosage of 5 to 10 units of heparin per milliliter of blood is placed into a syringe and the blood is collected.

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Smaller doses may be diluted with saline (250 units diluted in 2 rnL of saline per 50 rnL of blood is commonly used for cat transfusions). 34 Heparin is an effective anticoagulant both in vitro and in vivo, and is not recommended for routine collection and storage. Sodium citrate (3.8%) has also been used for blood collection at a ratio of 1 rnL sodium citrate to 9 rnL of blood. Citrate prevents coagulation by binding to calcium and thus inhibiting several calcium-dependent steps of the coagulation cascade. Sodium citrate has no preservative properties and is therefore recommended only for immediate-use transfusions, not for the storage of blood. Anticoagulant-Preservatives

The storage of blood using anticoagulant-preservatives or additive solutions is generally done in plastic bags rather than glass containers or syringes. Plastic bags do not readily break, facilitate the separation of blood components, avoid mechanical trauma to red blood cells when gravity collection is used, and are less likely to induce activation of platelets or coagulation factors. Also, during storage, gas exchange (carbon dioxide release and increased oxygenation) can take place through the wall of the plastic container. The container material for most blood or blood product storage is polyvinyl chloride (PVC) plasticized with di(2-ethylhexyl) phthalate (DEHP). DEHP migrates from the inner plastic surface into the stored blood and has a stabilizing effect on the red blood cell rnernbrane. 37 DEHP, however, has also exhibited carcinogenic properties in laboratory anirnals. 24• 50 Other less toxic plasticizers such as butyryl-n-trihexyl-citrate (BTHC) are now available in some commercial blood pack systerns. 39• 42 New generation plastic containers with increased gas permeability can be found in some multiple pack systems, where they are used to construct platelet storage bags for 5-day human platelet storage. 46 Examples of these new containers include those made from polyolefin without any plasticizer (PL-732, Fenwal Laboratories, Baxter Health Care, Corp., Deerfield, IL), PVC containers with a tri (2-ethylhexyl) trirnatallitate plasticizer (PL-1240, Fenwal Laboratories, Baxter Health Care, Corp., Deerfield, IL; and CLX Systems, Miles, Pharmaceutical Division, Elkhart, IN), or PVC containers plasticized with BTHC (PL-2209, Fenwal Laboratories, Baxter Health Care, Corp., Deerfield, IL). These newer containers are generally more expensive and are not necessary for routine canine or feline component storage. The composition of the traditional anticoagulant-preservative solutions used for whole blood or for component therapy are summarized in Table 2. After blood has been collected in these solutions, it can be stored as is until administered to patients, or the plasma can be removed and the packed red blood cells can be stored in a smaller amount of plasma (as is done with CPDA-1) or in a special additive solution (Table 3). All of these anticoagulant-preservative solutions contain certain cornpounds designed to maintain cell viability. Dextrose is present in suffi-

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Table 2. FORMULATION OF ANTICOAGULANT-PRESERVATIVE SOLUTIONS PRESENT IN BLOOD COLLECTION SETS Constituent

ACO-A

Volume (mL) Sodium chloride (mg) Dextrose (mg) Adenine (mg) Tri-sodium citrate (mg) Citric acid (mg) Sodium phosphate (monobasic) (mg)

67.5

ACO-B*

100

CPO

63

63

1610

2000 17.3 1660 206 140

None

None

None

None

None

None

1645

1485 540

None

1470

1320 480

None

CPOA-1

None

1660 206 140

ACD = acid-citrate-dextrose; CPD = citrate-phosphate-dextrose; CPDA-1 = citrate-phosphatedextrose-adenine. •Not available in a blood collection set but sold commercially as a solution (ACD Solution, USP, formula B, Fenwal Products, Baxter Healthcare Corp., Deerfield, IL}. Adapted from Wardrop KJ, Owen TJ, Meyers KM: Evaluation of an additive solution for preservation of canine red blood cells. J Vet Intern Med 8:254, 1994; with permission.

Table 3. FORMULATION OF ANTICOAGULANT-PRESERVATIVE AND ADDITIVE SOLUTIONS PRESENT IN BLOOD COLLECTION SETS Primary Preservative Constituent

Volume (mL) Sodium chloride (mg) Dextrose (mg) Adenine (mg) Mannitol (mg) Tri-sodium citrate (mg) Citric acid (mg) Sodium phosphate (monobasic) (mg)

CPO

63

None

1610

None None

1660 206 140

CP20

63 None

3220

None None

1660 206 140

Red Blood Cell Additive Adsol*

Nutricel

100 900 2200 27 750

100 410 1100 30

None None None

None

588 42 276

CPD = citrate-phosphate-dextrose. ·when Adsol is used, 450 ml of donor blood is first drawn into 63 ml of CPD. After centrifugation and removal of nearly all of the plasma, red blood cells are resuspended in 100 ml of the Adsol additive solution. When Nutricel is used, 450 ml of donor blood is drawn into 63 ml of CP2D, centrifuged, and the red blood cells are resuspended into 100 ml of the Nutricel additive solution after removal of the plasma. Adapted from Wardrop KJ, Young J, Wilson E: An in vitro evaluation of storage media for the preservation of canine packed red blood cells. Veterinary Clinical Pathology 23:84-86, 1994; with permission.

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cient quantity to support continuing ATP generation by glycolytic pathways. Adenine allows the cells to maintain their adenine nucleotide pool. Sodium citrate is the anticoagulant. Acid-Citrate-Dextrose

Acid-citrate-dextrose (ACD), also known as anticoagulant-citratedextrose, is a commercially available anticoagulant-preservative that has been used for the storage of red blood cells of both dogs and cats. ACD has been largely replaced by CPD or CPDA-1 for routine blood collection and storage, but is still used for small volume collection. The product is available in two formulations (see Table 1). ACD-formula "A" is used at a ratio of 1 mL ACD per 7 to 9 mL of blood. It is the anticoagulantpreservative most commonly used at the author's institution for whole blood, immediate-use feline transfusions (e.g., 6 mL ACD to 54 mL blood in a 60 mL syringe). ACD-formula "B" has a reduced citrate concentration and is used at a ratio of 1 mL ACD per 4 mL blood. Formula "B" is most often used for plasmapheresis, cytapheresis, and plasma exchange in humans. The 24-hour post-transfusion viability of feline red blood cells stored in ACD "B" is approximately 4 weeks, while the viability of canine cells in this same storage medium is approximately 3 weeks. 26, 41 Citrate-Phosphate-Dextrose

Citrate-phosphate-dextrose (CPD) maintains higher 2,3-DPG concentrations and a higher pH when used to store canine red blood cells than does ACDP It is most commonly used for the storage of canine or feline whole blood. Viability studies have not been performed using CPD in the dog and cat, but CPD-collected canine blood can be stored for 4 weeks before a significant decrease in oxygen release occurs. 31 Citrate-Phosphate-Dextrose-Adenine

Citrate-phosphate-dextrose-adenine (CPDA-1) is an anticoagulantpreservative solution available in commercially prepared multiple bag separation systems. It has thus become popular for use in preparation of canine (and less commonly feline) blood components. The added adenine in CPDA-1 provides a substrate from which RBCs can synthesize ATP, resulting in improved viability when compared to CPD without adenine. 1 CPDA-1 is generally used at a ratio of 1 mL CPDA-1 to 7 mL of blood. The recommended storage time for canine packed red blood cells in CPDA-1 is 20 days. 35 Whole blood may also be stored in CPDA-1. A recent study demonstrated that the 24-hour post-transfusion viability after 35 days of whole blood storage was approximately 82% for canine erythrocytes and 85% for feline erythrocytes using biotinylation labeling techniques. 3

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Additive Solutions

The newest generation of red blood cell preservatives to be evaluated by in vitro and in vivo techniques and used for both human and canine blood banking has been the additive solutionsY· 19- 21 These are protein-free solutions that are added to the red blood cells after plasma removal. In humans, the improvement in the red blood cell storage time obtained with these additive solutions is ascribed largely to their increased concentration of d extrose and adenine for red blood cell energy metabolism. Advantages of these additive sblutions include longer red blood cell storage, an increased yield of pl
Figure 2. Triple-pack blood collection set with citrate phosphate dextrose (CPO) and Adsol (Baxter Healthcare Corp. , Fenwal Division, Deerfield, IL) additive solution . Blood is collected into the primary bag (A) containing CPO and an attached 16-gauge needle. After centrifugation, the plasma is extracted into a satellite bag (B). The additive solution (C) is allowed then to flow into the primary bag and is mixed with the packed red blood cells. The empty additive solution bag may be used for the preparation of additional blood components.

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Baxter Health Care, Corp., Deerfield, IL), AS-3 (Nutricel, Miles, Pharmaceutical Division, West Haven, CT) and AS-5 (Optisol, Terumo Medical, Corp., Somerset, NJ) The additive solution Adsol can be successfully used to store canine packed red blood cells with a shelf-life of approximately 5 weeks (37 days). 48 The author has also recently demonstrated that the additive solution Nutricel is effective in the storage of canine red blood cells, with a 5-week shelf-life. 49

FUTURE DEVELOPMENTS New preservation media which prolong the shelf-life of red blood cells and improve red blood celL or plasma constituents continue to be developed and tested. A glucose, adenine, mannitol, citrate, phosphate, and ammonium chloride solution ("solution 6") was found in one study to maintain and even increase ATP levels in stored hump.n blood, providing acceptable in vivo survival values in human volunteers for up to 18 weeks. 28 Solutions using half-strength citrate improve the stability of factor VIII:C and decrease the loss of 2,3-DPG. 23 The use of in-line white blood cell reduction filters, available with some commercial blood pack systems, may reduce both red blood cell and platelet storage lesions, decrease the incidence of febrile nonhemolytic transfusion reactions, and reduce refractoriness to platelet transfusion. 33 Red blood cells have been frozen in special cryoprotectants for many years in the human field as a means of preserving rare red blood cell types, but this technique has not found use in the veterinary field because of the extensive post-thaw washing procedures involved. The use of cryoprotectants such as hydroxyethyl starch avoid these washing steps and have been experimentally studied in dogs. 43 Cell rejuvenation, a process involving the incubation of stored or previously frozen red blood cells in a special additive solution, has been performed using human cells and may someday find application in veterinary medicine. 9 • 27 Lyophilized red blood cells are also a future possibility, as new methods are demonstrating that red blood cells may be lyophilized in a manner that maintains their normal metabolic and enzymatic function upon rehydration. 14 All of these techniques have the potential for use in the veterinary field after appropriate study.

SUMMARY 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

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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. References 1. Akerblom OCH, Kreuger A: Studies on citrate-phosphate-dextrose (CPD) blood supplemented with adenine. Vox Sang 29:90, 1975 2. Beutler E, Wood L: The in vivo regeneration of red cell 2,3 diphosphoglyceric acid (DPG) after transfusion of stored blood. J Lab Clin Med 74:300, 1969 3. Bucheler J, Cotter SM. Storage of feline and canine whole blood in CPDA-1 and determination of the post-transfusion viability. J Vet Intern Med 8:172, 1994 4. Bunn HF, Briehl RW: The interaction of 2,3-diphosphoglycerate with various human hemoglobins. J Clin Invest 49:1088, 1970 5. Bunn HF: Differences in the interaction of 2,3-diphosphoglycerate with certain mammalian hemoglobins. Science 172:1049, 1971 6. Cavill I, Trevett D, Fisher J, et al: The measurement of the total volume of red cells in man: A nonradioactive approach using biotin. Br J Haematol 70:491, 1988 7. Chang JC, Van der Hoeven LH, Haddox CH: Glutathione reductase in the red blood cells. Ann Clin Lab Sci 8:23, 1978 8. Christian JA, Rebar AH, Boon GD, eta!: Senescence of canine biotinylated erythrocytes: Increased autologous immunoglobulin binding occurs on erythrocytes aged in vivo for 104 to 110 days. Blood 82:3469, 1993 9. Damus PS, Hicks M, Rosenberg RD: A generalized view of heparin's anticoagulant action. Nature 246:355, 1973 10. de Verdier CH, Garby L, Hjelm M, eta!: Adenine in red cell preservation: Posttransfusion viability and biochemical changes. Transfusion 4:331, 1964 11. Dumaswala UJ, Petrosky TL, Greenwalt TJ: Studies in red blood cell preservation: VI. Red cell membrane remodeling during rejuvenation. Vox Sang 63:12, 1992 12. Eisenbrandt DL, Smith JE: Evaluation of preservatives and containers for storage of canine blood. JAm Vet Med Assoc 163:988, 1973 13. Gabrio BW, Finch CA, Huennekens FM: Erythrocyte preservation: A topic in molecular biochemistry. Blood 11:103, 1956 14. Goodrich RP, Sowemimo-Coker SO, Zerez CR, et al: Preservation of metabolic activity in lyophilized human erythrocytes. Proc Nat! Acad Sci U S A 89:967, 1992 15. Greenwalt TJ, Dumaswala UJ: Effect of red cell age on vesiculation in vitro. Br J Haematol 68:465, 1988 16. Haradin AR, Weed RI, Reed CF: Changes in physical properties of stored erythrocytes. Relationship to survival in vivo. Transfusion 9:229, 1969 17. Heaton A, Miripol J, Aster R, et a!: Use of Adsol preservation solution for prolonged storage of low viscosity AS-1 red blood cells. Br J Haematol 57:467, 1984 18. Heaton WAL, Keegan T, Holme S, et a!: Evaluation of 99mTechnetiumf5 1Chromium posttransfusion recovery of red cells stored in saline, adenine, glucose, mannitol for 42 days. Vox Sang 57:37, 1989 19. Hogman CF, Hedlund K, Sahlestrom Y: Red cell preservation in protein-poor media. Vox Sang 41:274, 1981 20. Hogman CF, Hedlund K, Zetterstroem H: Clinical usefulness of red cells preserved in protein-poor mediums. N Eng! J Med 229:1377, 1978 21. Hogman CF, Akerblom 0, Hedlund K, et a!: Red cell suspensions in SAGM medium. Vox Sang 45:217, 1983 22. Hogman CF, de Verdier CH, Ericson A, et a!: Studies on the mechanism of human

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23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.

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Address reprint requests to K. Jane Wardrop, DVM, MS

Department of Veterinary Clinical Sciences College of Veterinary Medicine Washington State University Pullman, WA 99164-6610