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1 ARE YOU MY TYPE AND WHAT TO DO WHEN INCOMPATIBLE? Urs Giger, PD Dr. med. vet. Dipl. ACVIM & ECVIM-CA, Dipl. ECVCP Transfusion Center and Penn Animal Blood Bank, Section of Medical Genetics School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA Since the early 80s the use of blood products in treating critically ill animals and supporting animals undergoing surgical and other procedures has tremendously increased. However, it should be noted that blood products are prepared from donor animals and represent a very limited resource not available in all situations, and as they are biologicals they bear the inherent risks to transmit infectious agents and cause other adverse transfusion reactions. Furthermore, the need for blood typing and crossmatching of patients and donors has now been recognized in order to assure safe and more efficacious transfusions in dogs and cats. Veterinary clinicians play a key role in providing safe and effective transfusion therapy. To assure efficacious and safe transfusions blood donor and recipient should be blood typed and if previously transfused also crossmatched. Blood typing is clinically important to assure blood compatibility and therefore is recommended for any animal in need of a transfusion, considered to become a blood donor, and in cats prior to breeding to avoid hemolysis of the newborn kitten. Unless blood typing is regularly performed in practice it is best to send blood for typing to a laboratory. Different view points exist regarding the extent and methods used for compatibility testing and various techniques for laboratory or point-of-care use have been applied or are being developed. Dogs have more than a dozen blood group systems known as Dog Erythrocyte Antigens (DEA); there is no DEA 2. Canine erythrocytes are either positive or negative for a blood type e.g. DEA 4 positive or negative, and these blood types are thought be co-dominantly inherited. In the DEA 1 system, which represents an exception, DEA 1.1 (A1) and 1.2 (A2) are allelic and there may even be a DEA 1.3 (A3). 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. There are no clinically significant alloantibodies present prior to sensitization of a dog with a transfusion (not pregnancy). Table 1. Blood type frequencies in dogs (based upon limited surveys) Blood types % Positive % Negative DEA (A1) 1.2 (A2) * Antigen size in kd 50 & DEA 3 (B) DEA 4 (C) DEA 5 (D) NA DEA 7 (Tr) , 58 & 63 * DEA 1.1 and 1.2 negative dogs. NA: not available The clinically most significant canine blood type is DEA 1.1: DEA 1.1 (A1) elicits a strong alloantibody response after sensitization of a DEA 1.1 negative dog by a transfusion; thus can be responsible for a transfusion reaction in a DEA 1.1 negative dog previously transfused with DEA 1.1 positive blood. Transfusion reactions against other blood types have rarely been described. They include reactions against the DEA 4, Dal and another common red cell antigen, and other clinically important blood types may be found in the future. Only very limited surveys on the frequency of these blood types have been reported (Table 1), which suggest possible geographic and breed-associated differences. Some of the blood types are seen rarely (DEA 3), while others occur very commonly (DEA 4). In Japan additional blood group systems have been proposed, but their associations to the DEA systems and their clinical importance have not been documented. Recently, we have 675

2 identified an apparently new common red cell antigen that seems to be missing in some Dalmatians, hence named Dal red cell antigen. Strongly antigenic blood types are of great clinical importance, because they can elicit a potent alloantibody response. These alloantibodies may be of the IgG or IgM class and may be hemagglutinins or hemolysins. Clinically the most antigenic blood type in dogs appears to be DEA 1.1. Based upon experimental and clinical data, dogs can become sensitized after receiving a mismatched transfusion, i.e. a blood unit positive for one or more blood types not found on the recipient s red blood cells. Sensitizing dogs in experimental studies in the 1950 s led to the documentation of some transfusion reactions caused by blood group incompatibilities and to the characterization of new blood types. Canine blood typing is generally based on serologic identification by agglutination reactions. Originally serum from sensitized dogs has been used for typing, but such polyvalent alloantibodies vary from batch to batch and are therefore not optimal. Recently monoclonal antibodies against DEA 1.1 have been developed at Kansas State University and at the University of Lyon. Because of the strong antigenicity of DEA 1.1, typing of donors for DEA 1.1 is strongly recommended. Whenever possible, the recipient should also be typed to allow the use of DEA 1.1 positive blood for DEA 1.1 positive recipients. A blood typing card has been available for a decade (recently modified) as a simple standardized in-practice kit (DMS Laboratories, Flemington, NJ), in order to classify dogs as DEA 1.1 positive or negative. A cartridge has also become available from Alvedia DME (Lyon, France) which relies on a standardized simple chromatographic technique. Typing for DEA 1.1 is also available through most commercial and veterinary school laboratories. Furthermore, a unique gel column technology, widely used in human blood banking, has recently been found to be an excellent standardized laboratory method (DiaMed, Cressier, Switzerland). Caution should be exercised whenever the patient's blood is autoagglutinating or has a very low hematocrit (<10%). It is recommended to check for autoagglutination of blood with buffer/saline on a slide or the card. Autoagglutinating blood may be first washed three times with saline to overcome the apparent autoagglutination just like for the Coombs and crossmatch tests performed within tubes. However, if autoagglutination after three washes persists at >1+, it is considered to reflect true autoagglutination which precludes typing (as well as Coombs testing and crossmatching), because it will always look like DEA 1.1 positive blood. In such circumstances, DEA 1.1 negative blood should be used, until the patient does not agglutinate anymore and can be retyped. DEA 1.1 positive blood from very anemic animals may not agglutinate when exposed to the DEA 1.1 or other reagents because of the prozone effect. In these cases some of the patient's plasma may be discarded before applying a drop of blood onto the card. Finally, recently transfused dogs may display a mixed field reaction, with only the transfused or recipient cells agglutinating. Typing service and polyclonal antisera are available for DEA 1.1, 1.2, 3, 4, 5, and 7 (Animal Blood Resources International, Michigan), but their and other blood groups clinical importance has not been determined; they are identified by crossmatching previously transfused dogs. Use of these blood typing products, however, requires some expertise and experience. Some veterinarians recommend use exclusively of canine donors that are negative for all testable DEAs except DEA 4 (>98% of dogs are DEA 4 positive) in order to prevent sensitization against these blood types. However, we do not support the routine typing for other blood types then DEA 1.1 for several reasons: This protocol unnecessarily eliminates many active and potential donors. Based upon published frequencies less than 1 in 10 dogs would be acceptable. This extended blood typing protocol would be cost prohibitive because many dogs would need to be typed for every negative dog. Generally, humans are only typed for the ABO and Rh blood group system, although >2 dozen other blood group systems are known. Typing for more than DEA 1.1 does not eliminate the need for crossmatching following the first transfusion. Crossmatching may also identify incompatibilities against yet unknown types. There are no supporting published clinical reports that transfusion reactions could be substantially reduced by extended blood typing. The major feline blood group system thus far generally recognized is known as the feline AB blood group system and contains 3 alleles: type A, type B, and the extremely rare type AB. In contrast to dogs cats have naturallyoccurring alloantibodies which are responsible for transfusion reactions upon a first mismatched transfusion as well as neonatal isoerythrolysis in a first litter. More details about the AB and mik blood group system are summarized in the proceedings on Peculiarities of Feline Transfusion Medicine. 676

3 Blood type A and B frequency in cats in certain countries and breeds* Percentage (%) Percentage (%) Domestic shorthair cats Type A Type B Purebred cats Type A Type B USA Northeast Abyssinian North Central Am. shorthair Southeast Birman Southwest British shorthair West Coast Burmese Argentina Cornish rex Australia Devon rex India (Bombay) Exotic shorthair Europe Himalayan Austria Japanese Bobtail England Maine Coon 97 3 Finland Norwegian Forest 93 7 France Oriental shorthair Germany Persian Hungary Scottish Fold Italy Siamese Netherlands Somali Scotland Sphinx Switzerland Tonkinese Turkey Turk. Angora/Van *Ignoring the rare AB cats in many breeds with type B cats Whereas blood typing tests reveal the blood group antigens on the red blood cell surface, blood crossmatching tests assess the serologic compatibility or incompatibility between donor and recipient. Thus, the crossmatch test checks for the presence or absence of naturally-occurring and induced alloantibodies in serum (or plasma) without determining the blood type, and thus, does not replace blood typing. These antibodies may be hemolysins and/or hemagglutinins and can be directed against known blood groups or other red cell surface antigens. A standardized tube crossmatching procedure has been proposed and is used by many laboratories. The crossmatching test does require some technical expertise, may be accomplished through a veterinary laboratory along with blood typing and is done with EDTA-anticoagulated blood from recipient and potential donor. We have evaluated the novel DiaMed gel column technique and more recently the in-clinic DMS gel tube assay and found them in preliminary studies to be a promising simple, sensitive and standardized laboratory method to crossmatch dogs and cats. The major crossmatch tests for alloantibodies in the recipient's plasma against donor cells, whereas the minor crossmatch test looks for alloantibodies in the donor's plasma against the recipient's red blood cells. The presence of autoagglutination or severe hemolysis may preclude the crossmatch testing. A major crossmatch incompatibility is of greatest importance because it predicts that the transfused donor cells will be attacked by the patient's plasma, thereby causing a potentially life-threatening acute hemolytic transfusion reaction. As fatal reactions may occur with <1ml of incompatible blood, compatibility testing by administering a small amount of blood is not appropriate. This has been shown in experimental studies to result in fatal reactions. A minor crossmatch 677

4 incompatibility should not occur in dogs if canine donors have not been previously transfused and is of lesser concern because donor's plasma volume is small, particularly in packed red cell products, and will be markedly diluted in the patient. In contrast the major and minor crossmatch can show incompatibility prior to any transfusion due to the presence of naturally-occurring alloantibodies in cats, not only for the AB but also the Mik and possibly other blood group systems. The initial blood crossmatch between two dogs that have never before received a transfusion should be compatible, because dogs do not have naturally occurring alloantibodies. Therefore, one might omit a crossmatch before the first transfusion in clinical practice. Because the crossmatch does not determine the blood type of the patient and donor, a compatible crossmatch does not prevent sensitization of the patient against donor cells within 1 to 2 weeks. Therefore, previously transfused dogs and cats should always be crossmatched, even when receiving blood from the same donor. The time span between the initial transfusion and incompatibility reactions may be as short as 4 days and lasts for many years (i.e., years after the last transfusion alloantibodies may be present). Obviously, a blood donor should never have received a blood transfusion to avoid sensitization. In cats mixing a drop of donor/recipient blood with donor/recipient plasma will detect A-B incompatibilities if typing is not available. The practice of transfusing patients with the least compatible unit does not have any scientific basis. Nevertheless some minor agglutination results in crossmatching a patient may be unrelated to alloantibodies and unspecific (for instance patient s red cell damage by uremia and other illnesses, donor cells after extended storage). Of course any patient with true autoagglutination may not be matched. 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 (pyrogen-mediated fever, transmission of infectious agents, vomiting, mechanical hemolysis, congestive heart failure, hypothermia, citrate toxicity, pulmonary complications) and immunologic reactions (acute and delayed hemolytic transfusion reactions, urticaria to anaphylaxis, acute respiratory distress, graft versus host disease). Note that some clinical signs may be caused by both mechanisms. Despite the variety of blood types and the limited degree of compatibility testing in clinical practice, transfusion reactions are rarely reported. In surveys of blood product usage, transfusion reactions were observed in 2-13% of recipients, but none of them were definitely associated with blood group mismatches and hence could have been caused by other blood components or blood collection, processing and storage as well as host factors. Any transfusion of red cells or other components should be carefully monitored for its beneficial and adverse effects. A transfusion monitoring form should be with any patient receiving blood. EDTA blood samples from the patient prior to the transfusion and collection tube segments from the donor should be kept as well as preferably a small amount of the blood to be transfused from the unit. In case a transfusion reaction is suspected an incidence report form should be added and a work up should be performed. While the PCV increase can be estimated upon the volume of red cells administered and size of the animal, the actually achieved results in a particular patient varies and should be assessed by direct PCV measurements 1 hour and 12 and 24 hours after the transfusion. In critically ill patients fluid shifts and saline and other infusions can also diminish the PCV rise just like continues blood and fluid losses. Severe hemolytic transfusion reactions due to blood type incompatibilities may cause anaphylaxis and acute death, but classically result in hemoglobinemia and hemoglobinuria, and if time permits icterus and hyperbilirubinuria. Bradycardia, tachycardia or arrhythmia, fever, and hypotension may be noted. While in humans acute renal failure due to free hemoglobin is a major consequence of incompatible transfusions, this appears not to be a problem in dogs and cats. Finally, a lack of a PCV rise or rapid drop in PCV following the transfusion may be the only indication of a mild or delayed hemolytic transfusion reaction. At the first sign of any transfusion reaction the blood component administered should be stopped. It is imperative to provide supportive care realizing there is no specific effective therapy besides discontinuing the transfusion to prevent further harm. Management with fluids, antihistamines, and glucocorticoids is often initiated without scientific evidence of its efficacy. Any transfusion reaction should be immediately and carefully investigated. In case of hemolysis the patient s and donor s blood type should be confirmed by review of records and retyping, and a crossmatch between patient and donor blood should be performed. If an incompatibility is recognized extended 678

5 typing against known DEA and dal blood types may be performed in dogs and mik type in cats. Incompatible blood should never be administered. A search for a compatible blood among screened donors should be initiated. Transfusion should not be restarted until after a thorough investigation has been concluded that assures the blood quality and compatibility to safely administer blood. Specific references are available upon request. 679

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