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Veterinarian Technician May 2013 (Vol 34, No 5)

Clinical Pathology Close-up: Blood Typing

by Margi Sirois, EdD, MS, RVT

    CETEST This course is approved for 1.0 CE credits

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    The increased availability of blood components such as packed red blood cells (pRBCs) and platelet-rich plasma has improved treatment for some patients in emergency and critical care settings. Veterinary blood banks provide blood components, and most also perform blood typing and crossmatching. These procedures can also be performed in-house in veterinary practice laboratories. Veterinary technicians must understand the concepts of blood component transfusion and the procedures to help ensure that transfusion therapy is safe.

    Blood Groups and Immunity

    Red blood cell (RBC) antigens are structures on RBC surfaces in an animal that may react with antibodies in the plasma of another animal. The specific surface markers in an individual animal are genetically determined and are referred to as blood group antigens. The number of blood groups varies among species. Antigen-antibody reactions can occur with blood transfusions due to variation in blood group antigens between the recipient and the donor. These reactions usually results in clumping or agglutination of RBCs or may manifest clinically as RBC lysis.

    The erythrocytes of some domestic animals (i.e., cats, cattle, sheep, and pigs) have naturally occurring antibodies (alloantibodies). Once a transfusion has been given to an animal, antibodies against the RBC antigen (immune antibodies) of the transfused blood form. Breeding females should always be given properly matched blood to minimize the potential for production of antibodies that can result in destruction of a neonate’s RBCs.1

    Blood Types

    Dogs

    More than a dozen different canine blood groups have been described. Nomenclature for the blood group systems is designated by DEA (dog erythrocyte antigen) followed by a number. For DEA systems other than DEA 1, the erythrocytes are designated as positive or negative for the specific antigen. The DEA 1 group comprises subgroups DEA 1.1, DEA 1.2, and DEA 1.3. Canine erythrocytes may be positive or negative for each of the DEA 1 subgroups. DEA 3, DEA 4, DEA 5, and DEA 7 designate other major blood groups.2 The blood groups considered to be clinically significant are DEA 1.1 and DEA 7. DEA 1.1 elicits the greatest antigen response and causes the most serious transfusion reactions. Approximately 50% of dogs are positive for DEA 1.1. Transfusion reactions to the other blood groups are less likely to cause clinical signs. Dal, another canine antigen, has also been described.3 Because naturally occurring anti–DEA 1.1 antibodies are not known to exist, the first transfusion of DEA 1.1–positive blood into a DEA 1.1–negative recipient may not result in an immediate reaction. However, antibodies can develop, resulting in a delayed transfusion reaction in as little as a week after the original mismatched transfusion. If a DEA 1.1–negative dog previously received DEA 1.1–positive blood, a severe reaction can occur in less than 1 hour if the dog is subsequently transfused with DEA 1.1–positive blood.2

    Cats

    One blood group system, designated the AB system, has been identified in cats. The blood groups of cats are A, B, and AB. Few cats have group AB blood. The vast majority of cats in the United States have group A blood, which probably accounts for the low incidence of transfusion reactions in cats. Type B blood is found in certain breeds (e.g., Devon rex, British shorthair) and certain geographic areas (e.g., Australia). Unlike dogs, cats have naturally occurring antibodies to the erythrocyte antigen they lack. Type B cats have strong anti-A antibodies, while type A cats have weak anti-B antibodies. Transfusing type B cats with type A blood may result in a serious transfusion reaction and death. Thus, blood for transfusion of purebred cats should be selected by typing or crossmatching. Mik, another blood cell antigen, has also been described in cats.4

    Neonatal isoerythrolysis has been documented in type A and type AB kittens of type B queens with naturally occurring anti-A antibodies.2

    Blood Typing

    Methods of identifying some canine and feline blood groups are available for use in veterinary practice. These methods include an immunochromatography assay and a card/slide agglutination assay.4 The tube method is the gold standard for blood typing but is primarily used in reference laboratories.

    The Tube Method

    The tube method for determining blood type requires the use of antisera, which consist of antibodies specific for each possible blood type of a given species. Commercial antisera for canine and feline group testing are available for a few canine and feline blood groups (BOX 1). The tube method requires collection of a whole blood sample using EDTA, heparin, or acid-citrate-dextrose anticoagulant. The blood is centrifuged at 1000g for 10 minutes. After removal of the plasma and buffy coat, the erythrocytes are washed three times in a saline solution, centrifuged, and resuspended. The RBC suspension is distributed among as many tubes as required for the number of blood type antisera being tested. A small amount (usually 0.1 mL) of the antisera is added to the appropriately labeled tube. The tubes are incubated for 15 minutes at room temperature and then recentrifuged for 15 seconds at 1000g. Each tube is examined macroscopically and microscopically for evidence of hemolysis or agglutination. Weak positive results may require repeat testing.2

    The Card Agglutination Test

    Blood samples used to perform the card-based assay must not already show evidence of autoagglutination, which is usually visible as clumps in the blood sample. Washing the RBCs with phosphate-buffered saline may help salvage a sample that is showing evidence of agglutination. The RapidVet-H Canine DEA 1.1 (DMS Laboratories) is a blood-typing test card used to classify dogs as positive or negative for DEA 1.1 (FIGURE 1). The typing card contains a monoclonal antibody specific to DEA 1.1. Each card has three visually defined wells labeled “Autoagglutination patient screen,” “DEA 1.1–positive control,” and “Patient test.” One drop of EDTA-anticoagulated whole blood and 1 drop of phosphate-buffered saline are mixed on the lyophilized reagents within each well. In the DEA 1.1–positive test well, the monoclonal antibody forms an antiserum and is then mixed with whole blood from the patient. If present, DEA 1.1–positive erythrocytes react with the antiserum, causing agglutination. The antiserum in the patient test well does not react with DEA–1.1 negative erythrocytes.

    RapidVet-H (feline) is a similar blood-typing test card for classifying feline blood as type A, B, or AB. The assay uses test wells that contain lyophilized reagent, representing an antibody to the A antigen, and an anti-B antigen, consisting of a lectin. Erythrocytes from type A cats agglutinate with anti-A monoclonal antibodies (the A well on the card), and erythrocytes from type B cats agglutinate with anti-B solution (the B well on the card). Erythrocytes from type AB cats agglutinate with both anti-A and anti-B reagents. The third well on the card serves as an autoagglutination saline screen and must be negative for results to be valid. Samples are first screened for autoagglutination. If autoagglutination is present, the RBCs may be washed with phosphate-buffered saline and the autoagglutination screen repeated. If the autoagglutination result is negative, the typing test may be performed.5

    Immunochromatographic Assay

    Two commercial test kits use the immunochromatographic test principle rather than agglutination. The control band detects a separate antigen on the RBCs. The canine test uses a paper strip impregnated with monoclonal anti–DEA 1.1 antibody and a second antibody to a universal RBC antigen as a control. An RBC solution diffuses up the strip, and if the cells express DEA 1.1, they concentrate in the area of antibody impregnation. The cells also concentrate in the area of the control antigen, demonstrating that cells have successfully diffused up the length of the strip. The feline test works the same way; however, it has an area containing an anti-A monoclonal antibody, an area containing an anti-B monoclonal antibody, and a control antibody for a common feline RBC antigen, allowing identification of blood type A, B, or AB6 (FIGURE 2).

    Crossmatching

    In the absence of commercial antisera, crossmatching a blood donor and a recipient reduces the possibility of a transfusion reaction.7 The two-part procedure (major and minor crossmatches) requires a serum sample and a whole blood sample (BOX 2). RBC suspensions, collected as for blood typing, are prepared. In the major crossmatch, a few drops of serum from the recipient are added to a few drops of washed pRBCs from the donor. The mixture is incubated and then centrifuged. Macroscopic or microscopic presence of hemolysis or agglutination indicates a blood-type mismatch. The minor crossmatch is similar except that donor serum and recipient RBCs are used. Both procedures should be performed on all animals that require transfusion but whose blood types are unknown. Two controls are used for the test, which consists of using donor cells with donor serum as well as recipient cells with recipient serum. A commercial test kit for crossmatching is also available (FIGURE 3).

    Agglutination reactions are sometimes graded. Several classification schemes are used for this purpose. TABLE 1 shows one type of grading scale. The clinician determines whether evidence of agglutination constitutes an unsuitable transfusion.

    Summary

    Proper blood typing and crossmatching can provide valuable information to clinicians and help minimize problems in critically ill patients. Ideally, all critically ill patients should undergo blood typing and crossmatching before a transfusion.

    Downloadable PDF

    Dr. Sirois is section editor of Veterinary Technician's Clinical Pathology Close-up series.

    1. Giger U. Transfusion medicine. In: Silverstein D, Hopper K, eds. Small Animal Critical Care Medicine. St. Louis, MO: WB Saunders; 2009:282-286.

    2. Hill E. Immunology, serology, and molecular diagnostics. In: Sirois M, Hendrix C, eds. Laboratory Procedures for Veterinary Technicians. 5th ed. St. Louis, MO: Mosby Elsevier Health Science; 2007:264-286.

    3. Blais MC, Berman L, Oakley DA, et al. Canine Dal blood type: a red cell antigen lacking in some Dalmatians. J Vet Intern Med 2007;21(2):281-286.

    4. Weinstein NM, Blais MC, Harris K, et al. A newly recognized blood group in domestic shorthair cats: the Mik red cell antigen. J Vet Intern Med 2007;21(2):287-292.

    5. Hohenhaus A. Blood transfusion and blood substitutes. In: DiBartola SP, ed. Fluid, Electrolyte, and Acid-Base Disorders in Small Animal Practice. 4th ed. St. Louis, MO: Elsevier; 2012:593-594.

    6. Oakley D, Calabrese M. Fluid therapy and blood transfusion diagnostics. In: Sirois M, Hendrix C, eds. Laboratory Procedures for Veterinary Technicians. 5th ed. St. Louis, MO: Mosby Elsevier Health Science; 2007:434-446.

    7. Blood groups and blood transfusions. In: The Merck Veterinary Manual. 9th ed. Accessed January 2013 at http://www.merckvetmanual.com/mvm/index.jsp?cfile=htm/bc/toc_10300.htm.

    References »

    NEXT: Final View: Buddy’s Binge

    CETEST This course is approved for 1.0 CE credits

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