Urs Giger, PD Dr. med. vet. MS FVH, DACVIM, DECVIM
Blood Types and Alloantibodies
Blood types are genetic markers on erythrocyte surfaces which are antigenic and specific for each species. A set of allelic blood types (two to several alleles) makes up a blood group system. Blood types can be identified in vitro by hemagglutination and/or hemolysis reactions using species-specific antisera against animal red blood cells. Animals may have or develop antibodies against the other blood type(s) of a blood group system, known as allo- or isoantibodies. Naturally-occurring alloantibodies are found in the sera of unimmunized animals, whereas other alloantibodies occur only after sensitization by a transfusion, prior pregnancy, or administration of animal tissue-containing products (vaccines). Blood types against which naturally-occurring alloantibodies exist or alloantibodies are readily induced are of great clinical importance.
Canine blood typesDogs have at least thirteen blood types known as Dog Erythrocyte Antigens (DEA) 1 through 13 (no DEA 2) and represent separate blood group systems. Thus, a dog can be DEA 1.1 positive or negative and DEA 1.1 negative dogs can be DEA 1.2 positive or negative. Dog’s erythrocytes are either positive or negative for a blood type e.g. DEA 4 positive or negative. In the DEA 1 system (exception) DEA 1.1 (A1) and 1.2 (A2) are allelic. There are no clinically significant alloantibodies present prior to sensitization of a dog. The frequency of each blood type may vary considerably between canine breeds.
Blood Type Frequencies in Dogs (*DEA 1.1 and 1.2 negative dogs)
Clinically significant canine blood types are
Simple blood typing cards utilizing a DEA 1.1 monoclonal antibody are available for DEA 1.1 typing of dogs (DMS laboratories, New Jersey) and other clinical typing procedures are being evaluated. It is recomm
Feline blood types
The feline AB blood group system contains 3 alleles: A, B, and the extremely rare AB. Type A is dominant over B. Thus, cats with type A blood have the genotype A/A or A/B, and only homozygous B/B cats express the type B antigen on their erythrocytes. In the extremely rare AB cat, a third allele recessive to A and dominant to B leads to the expression of both A and B substances. AB cats are not produced by mating a type A to a type B cat unless the A cat carries the rare AB allele. Cats with type AB blood have been seen in many breeds and domestic shorthair cats (mostly West Coast).
Blood type A and B frequency in cats (*breeds with isolated type AB cats)
The frequency of feline A and B blood types varies geographically and between breeds. Most domestic shorthair cats have type A blood. The frequency of A and B blood types of domestic shorthair cats differs geographically. The frequency of A and B blood types varies greatly between different breeds. Kitten losses due to A-B incompatibility and changes in breeding practices influence the frequency of A and B in various breeds. Nearly all blood donors have type A blood. They must be typed.
Cats have naturally-occurring alloantibodies. All type B cats have very strong naturally-occurring anti-A alloantibodies. Kittens receive alloantibodies through the colostrum from type B queens and develop high alloantibody titers (>1:32) after a few weeks of age. These alloantibodies are strong hemolysins and hemagglutinins, and are of the IgM and, to a lesser extent, IgG classes. They are responsible for serious transfusion reactions and neonatal isoerythrolysis. Type A cats have weak anti-B alloantibodies. The alloantibody titer is usually very low (1:2) and can be detected by hemolysis and hemagglutination assays. They cause shortened survival of transfused B cells in type A cats, but have not been associated with neonatal isoerythrolysis. Type AB cats have no alloantibodies.
There are no universal donor cats. Donor and patient need to be typed. Simple blood typing cards are available for in practice use (DMS Laboratories, New Jersey). Other practical typing procedures are being developed. Feline blood typing typing service are available from Dr. Urs Giger, Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104-6010.
Blood crossmatch test detects the serologic (in-) compatibility between the anemic recipient and potential donor, and should be performed prior to any transfusion. Thus, this test looks for the presence or absence of allo-antibodies without determining the blood type. The crossmatching test requires anticoagulated blood from recipient and potential donor.
Blood type of canine donor: Ideally donor and recipient should have identical blood types. All canine blood donors should be blood typed. Canine blood donors should be DEA 1.1 (A1) negative unless the recipient is known to be positive. DEA 1.1 negative dogs are considered universal donors. DEA 1.1 positive can receive DEA 1.1 positive blood. (Non-typed) recipients should be crossmatched with potential donors to determine compatibility after initial transfusion. Dogs not previously transfused will generally have a compatible crossmatch and develop no signs of an acute hemolytic transfusion reaction because they lack significant naturally-occurring allo-antibodies.
Blood type of feline donor: There are no universal feline blood donors because cats have naturally-occurring alloantibodies. A-B mismatched transfusions can result in life-threatening transfusion reactions. All feline blood donors and recipients should be blood typed. If because of time constraints blood typing cannot be performed, potential donor and recipient have to be cross-matched. Most feline blood donors are domestic shorthair cats and have type A blood. Type B blood donors are available at some institutions and blood banks. A type B blood donor may be easily identified by typing clients' purebred cats. The extremely rare AB cat can be safely transfused with type A blood (preferably type A packed red cells), if type AB blood is not available.
Preparation of donor: Donor compatibility with recipient has been determined if blood is specifically drawn for a particular patient. Packed cell volume or hemoglobin and body weight are obtained, a physical examination is performed, and all of them are compared to the data recorded last time. If there is any concern regarding the health status of the donor, no blood should be collected. Canine donors do not have to be tranquilized for collection. Feline donors are commonly sedated with a combination of ketamine 10 mg, diazepam 0.5 mg, and atropine 0.04 mg by intravenous injection. Some sedatives, such as acepromazine, interfere with platelet function and induce hypotension. Animals that are exsanguinated will be anesthetized with barbiturates. Aseptic techniques are used to collect blood by jugular vein catheterization. Detailed records should be kept for each donor animal regarding blood type, dates and data of health examinations, and dates of blood withdrawal and amount of blood drawn. Any transfusion reactions observed after blood from a donor is administered should be recorded.
Blood is collected aseptically by gravity or vacuum pump from the jugular vein. Plastic bags (Fenwal) containing citrate-phosphate-dextrose-adenine (CPD-A1) with or without satellite bags for blood component separation are optimal. This represents a closed collection system in which the blood does not come into contact with the environment at any time during collection or separation into blood components, thus minimizing the risk of bacterial contamination and allowing storage of the blood products. Large plastic syringe containing 1 ml CPD-A1 or 3.8% citrate per 9 ml blood and connected to a 19 gauge butterfly needle is commonly used for cats. This represents an open collection system in which connections allow exposure of blood to the environment; because of the potential risk for bacterial contamination, blood collected via an open system should not be stored for more than 48 hours. A closed blood collection system for cats has been developed that allows blood component preparation and storage. Vacuum glass bottles containing acid-citrate-dextrose (ACD) allow rapid collection but are not recommended because blood components are readily damaged and cannot be separated and stored for long.
Anticoagulants and preservatives used include 3.8% sodium citrate which is the best anticoagulant solution. Citrate alone (i.e., no preservatives) can be used to collect blood for immediate transfusion. ACD, CPD or CPD-A1 are commercial anticoagulant preservative solutions that should provide adequate red cell function and viability. Viability of red cells depends on the ability to generate ATP through anaerobic glycolysis. Dextrose, phosphate and adenine are substrates for ATP production, which proceeds slowly at cold temperature. In canine erythrocytes, the hemoglobin-oxygen affinity is greatly dependent on the amount of 2,3-diphosphoglycerate (DPG). High erythrocyte DPG concentrations facilitate the release of oxygen from hemoglobin to tissue. CPD and CPD-A1 are superior to ACD in preserving canine erythrocyte DPG concentrations. Erythrocyte DPG levels are restored within 24 hours after transfusion. Viable red cells will quickly regenerate erythrocyte DPG after transfusion. CPD-A1 is most commonly used to store whole blood, packed red cells, and plasma components. Feline erythrocytes have very low DPG concentrations. DPG is not usually required for adequate oxygen release from feline hemoglobin. Thus, ACD and CPD may be comparable in preserving feline red cells. Heparin (15 IU/ml blood) is not recommended as an anticoagulant solution because it may induce platelet aggregation and inhibits many coagulation factors. Heparin cannot be used to store blood. Maximal blood volume to be donated:
Separation and Storage of Blood Components
The separation of blood components from a single donation of blood is performed according to the Technical Manual of the American Association of Blood Banking and does require some expertise and equipment. Blood component separation may also be performed at a nearby human hospital. Blood component preparation is best accomplished by using plastic blood bags with satellite transfer containers in order to assure sterility. Blood components are prepared within 8 hours from the time of collection except for stored (frozen) plasma which can be separated from red cells anytime until 4 weeks after collection. Transfer bags may also be used to make smaller blood units (e.g., 100 ml) for smaller dogs. Pediatric transfer packs are utilized by PABB for a closed feline blood collection system; feline blood may be stored as whole blood or separated into components (as described below). Fluctuations in storage temperature significantly alter the length of storage; thus, temperature controlled and alarmed blood bank refrigerators and freezers are ideal, but others are acceptable as long as the temperature is monitored and the refrigerator/freezer are not too frequently opened. Blood components that have been warmed to room or body temperature should not be recooled and cannot be stored again. Similarly, partially used or opened blood bags should be used within 24 hours, because of the risk of contamination. Indications for blood component therapy are summarized:
INDICATIONS FOR BLOOD COMPONENT THERAPY
(◙) indicates best component; FWB = fresh whole blood, SWB = stored whole blood, PRBC = packed red blood cells, PRP = platelet-rich plasma, PC = platelet concentrate, FFP = fresh frozen plasma, CRYO = cryoprecipitate
Fresh whole blood (FWB): By definition, this blood product has not been stored. After collection, the blood is usually kept at room temperature with gentle agitation for less than 8 hours prior to transfusing a patient. In FWB, all blood components (RBCs, platelets, coagulation factors, plasma proteins) are present and functional. FWB is indicated in anemic patients with thrombocytopenia/thrombopathia causing severe, uncontrolled or life-threatening bleeding.
Stored whole blood (SWB): Whole blood can be stored in a refrigerator at 4oC for approximately one month when collected in ACD, CPD or CPD-A1. SWB contains RBCs and plasma proteins (albumin and globulins), but not functional platelets or labile coagulation factors. SWB is indicated in anemic animals with hypoproteinemia (e.g., severe iron deficiency anemia due to chronic external blood loss from endo-/ectoparasitism).
Packed red blood cells (PRBCs): PRBCs are separated from plasma by centrifugation at 4oC. One unit of blood will result in approximately 200-250 ml plasma and 250-300 ml PRBCs with a PCV between 70 and 80%. PRBCs can be stored at 4oC for approximately one month. A special preservative solution (e.g., ADSOL – adenine, dextrose, saline, and mannitol) may be added to the PRBCs from a satellite bag to extend the storage time to 35 days. If PRBCs do not contain ADSOL, physiologic saline (100 mls) may be added to decrease the viscosity (PCV) of the PRBCs prior to transfusion. Do not substitute physiologic saline with any other solution. PRBCs are the blood component of choice for severely anemic animals to provide additional oxygen-carrying support.
Fresh frozen plasma (FFP): FFP is separated from PRBCs and frozen within 8 hours from collection of blood. FFP can be stored at -30oC or less for up to one year. FFP contains coagulation factors and plasma proteins; plasma that is refrozen after thawing loses its labile coagulation factors. FFP is indicated in the treatment of coagulopathies (e.g., hereditary coagulopathies, liver disease, anticoagulant rodenticide poisoning) and von Willebrand’s disease (if cryoprecipitate is not available).
In cats, the blood type of plasma should be labeled due to the presence of naturally occurring alloantibodies against the other blood type. In dogs, it is also preferable to label the plasma according to the blood type of the donor (i.e., DEA 1.1-positive or DEA 1.1-negative) since the presence of a small number of RBCs in the plasma may lead to sensitization of the recipient.
Platelet-rich plasma (PRP) and platelet concentrate (PC): PRP is prepared from fresh whole blood (“light spin” at room temperature); the PRP can undergo further centrifugation to form a platelet concentrate, and the supernatant plasma removed and stored as FFP. PRP and PC can be stored at room temperature under constant gentle agitation for 48 hours. However, platelet products are usually used immediately after their preparation. The platelet count of PRP or PC is approximately 3 x 1011/unit (unit defined as platelets derived from 1 unit (450 ml) of FWB). PRP or PC is indicated in thrombocytopenic/thrombopathic animals with uncontrollable, severe or life-threatening bleeding (e.g, intracranial hemorrhage).
Cryoprecipitate (CRYO): CRYO is prepared from fresh frozen plasma within 12 months of collection by slowly thawing the plasma at 4oC (refrigerator) overnight. When 10% of the content is still frozen, the product is centrifuged and the liquid plasma then transferred into another satellite bag with the plasma extractor. CRYO will stick to plastic surface. Both CRYO and remaining CRYO-poor plasma are immediately refrozen. CRYO contains von Willebrand factor, factor VIII, fibrinogen, and fibronectin and is mainly indicated in management of bleeding due to von Willebrand’s disease and hemophilia A. CRYO-poor plasma may be used for other coagulopathies or for colloidal support in hypoproteinemic patients.
Administration of Blood
For routine transfusion in the treatment of anemia, it is not necessary to warm blood after removal from the refrigerator. Warming may in fact accelerate the deterioration of stored red blood cells and permit rapid growth of contaminating microorganisms. However, there are specific clinical situations such as transfusion of neonates or resuscitation of trauma patients necessitating rapid, massive transfusions, in which warming of blood is indicated to prevent complications associated with hypothermia (e.g., cardiac arrhythmias and coagulopathies). A temperature controlled waterbath (#37oC) is ideal to warm blood products. A warm water bowl in which the water is periodically changed may be used to warm blood products. Care should be taken to maintain absolute sterility and to not overheat the blood products. Blood components that have been prewarmed cannot be refrozen/refrigerated.
Blood bags are connected to blood infusion sets that have an in-line microfilter. A long (85 cm) blood infusion set with a dripping chamber and a short infusion set for small dogs and cats to connect with syringes are available. Use a latex-free infusion set for platelet administration to avoid aggregation. Microfilter with 170 μm pores are commonly used to remove clots and larger red cell and platelet aggregates. Finer filters with 40 μm pores will remove most platelets and microaggregates. Leukocyte reduction filters (expensive) may be used to decrease adverse reactions to WBC components. Febrile transfusion reactions have been attributed to release of pyrogens (e.g., IL-6, IL-1β) from WBCs during storage of blood and from reactions between donor leukocytes and recipient antibodies. Sterility has to be maintained when connecting bag to infusion set and tubing to catheter. The infusion set may be directly locked onto the catheter hub.
Blood components are best administered intravenously. Ideally, an indwelling catheter (16-22 gauge depending on size of animal) is placed into the jugular vein but cephalic or saphenous vein on extremities may also be used. In case an intravenous access cannot be obtained, red blood cells and plasma may be administered by intramedullary (or intraosseous) infusion at the trochanteric fossa (or other site). A standard needle, spinal needle, or bone marrow needle can be used ,depending on the age and size of the animal. Approximately 80-95% of cells will be in circulation minutes after transfusion. Avoid concurrent administration of drugs or fluids other than physiologic saline through the same catheter in order to prevent lysis of erythrocytes and blood coagulation. Thus, do not administer calcium or glucose containing or hypo- or hypertonic solutions through the same IV line.
Rate of transfusion depends on the hydration status, degree of anemia, and general health condition of an animal. Initial rate is slow, starting with 1-3 ml over the first 5 minutes to observe for any transfusion reaction, even with blood typed or matched transfusions. In animals with cardiac failure, do not exceed 4 ml/kg/hr. Transfusion of a single bag should be completed within 4 hours to prevent functional loss of blood elements or bacterial growth.
Volume of blood component to be administered depends on the degree of anemia, platelet defect or coagulopathy, and the size of the animal.
In anemia: Volume (ml) of whole blood = PCV rise desired (%) x body weight(lbs), or in other words,
administration of 1 ml whole blood/lb body weight elevates the PCV by 1%.
PCV rise desired is the aim for PCV after transfusion minus the recipient's actual PCV; this formula assumes that the PCV of the blood bag is 40%. If packed red cells are used, half the volume has to be administered, since packed red cells have a PCV of 70-80%. In the absence of bleeding and hemolysis, at least 70% of transfused erythrocytes survive 24 hours (required blood bank standard) and transfused erythrocytes may be thereafter expected to have a normal life-span (approximately 70 days in cats, 110 days in dogs). Monitor response to transfusion by obtaining PCV/TP readings prior to, immediately, and 6 and 24 hours post-transfusion, and consider continued blood loss and/or hemolysis.
In thrombocytopenia or thrombopathia, one unit of PC, PRP or FWB will increase the platelet count by 10,000/μL in recipient weighing 30 kg. In animals with serious or life-threatening bleeding, the platelet count should be increased to above 40,000/μL. Platelet counts are monitored prior, 1 hour and 24 hours after the platelet transfusion. In immune-mediated thrombocytopenia, the survival of transfused platelets is extremely short (min). Platelet transfusions may have to be repeated daily to maintain platelet counts above 40,000/μL and to avoid rebleeding.
In coagulopathies and von Willebrand’s disease, FFP: 6-10 ml/kg is an initial dose to stop bleeding or avoid excessive bleeding during surgery. In some cases, larger volumes may be needed to control bleeding. Depending on the coagulopathy, administration of FFP may require repeated administration. Because of the short half-life of factor VII and VIII and von Willebrand factor, deficient animals need to be treated twice daily. Other coagulopathies may be treated daily. Cryoprecipitate at a dose of 1 cryo unit/10 kg or 1-2 ml/kg body weight twice daily is ideal to treat Hemophilia A and von Willebrand’s disease. Plasma support should be provided for an additional 1-3 days after the bleeding has been controlled to allow for healing and prevent rebleeding.
Complications of Transfusions
While transfusion of blood and its components is usually a safe and temporarily effective form of therapy, there is always a risk for potential hazards. Adverse reactions usually occur during or shortly after the transfusion and can be due to any component of whole blood. Most transfusion reactions can be avoided by carefully selecting only healthy donors, using appropriate collection, storage, and administration techniques, performing blood typing and crossmatching, and administering only needed blood components. The most common clinical sign of transfusion reaction is fever, followed by vomiting and hemolysis. Hemolytic transfusion reactions can be fatal and are, therefore, most important, while fever and vomiting are usually self-limiting. Adverse effects of transfusions can be divided into non-immunologic and immunologic reactions. Note that some clinical signs may be caused by both mechanisms.
ended to blood type patient and donor for DEA 1.1. Dogs that are DEA 1.1 negative are considered universal blood donors. Canine blood typing sera for DEA 1.1, 1.2, 4 and 7 and typing services are available from Midwest Blood Services, East Lansing, MI. Different blood group systems based upon monoclonal blood typing reagents are being proposed by Japanese investigators(Shigeta, Japan).
ADVERSE REACTIONS TO TRANSFUSIONS
Giger Urs (1997), “Transfusion Medicine,” in Handbook of Small Animal Practice, R.V. Morgan, ed. Philadelphia, PA, WB Saunders Co.
Dr. Urs Giger, Veterinary Hospital University of Pennsylvania, 3850 Spruce
Street, Philadelphia, PA 19104-6010. 215 898 8894; 215 898
3375 (lab); fax 215 573 2162;