PT8

Type & Cross-Match in Practice Michael J. Day, BSc, BVMS(Hons), PhD, FASM, DECVP, MRCPath, FRCVS School of Clinical Veterinary Science, University of Bristol UK
OBJECTIVES OF THE PRESENTATION

To review the canine and feline blood group systems. To discuss the basis for blood transfusion reactions. To describe methodology for in-house blood typing of cats and dogs. To describe the methodology for in-practice cross-matching using the tube or slide technique.
KEY POINTS

The canine and feline blood group antigen systems differ. Incompatibility in blood group antigens and the presence of spontaneously arising or induced alloantibodies may lead to blood transfusion reactions. Pre-transfusion blood typing and cross-matching is best clinical practice in all cases. Blood typing and cross-matching may be performed by commercial laboratories or in the practice laboratory.
OVERVIEW OF THE ISSUE Introduction

Blood transfusion is increasingly performed in companion animal medicine. In the USA blood banking facilities and the use of blood components or bovine haemoglobin is commonplace, but in Europe fresh whole blood transfusion is still normal practice. This presentation considers the practice of whole blood transfusion. The most important consideration for safe blood transfusion is avoidance of transfusion reactions due to administration of incompatible donor blood. Transfusion reactions may arise with an incompatible first transfusion in the cat, but in general only occur on second or subsequent transfusion in the dog.
Blood Groups in Dogs and Cats

Although in theory, multiple components of a whole blood transfusion might lead to an adverse reaction with an incompatible donor-recipient pairing, it is incompatibility in the erythrocyte blood group antigens that is responsible for most transfusion reactions. The canine blood group antigen system is relatively complex and includes at least 10 blood group antigens1. Fortunately, in practical terms only two of these (DEA 1.1, DEA 1.2) are of clinical significance. In general terms, dogs may either express DEA1 antigens or not. Those dogs that do not carry DEA1, do not have a spontaneous alloantibody to this blood group, but such antibodies can be induced, most commonly by administering DEA1 positive blood to a DEA1 negative dog or sometimes following pregnancy. Dogs negative for the DEA3, 5 or 7 antigens may rarely have low titred alloantibodies against these molecules. In practical terms, the majority of dogs do express DEA1 blood group antigens which minimises the risk of adverse reactions. The old adage that a first transfusion is safe in the dog is probably correct, but good clinical practice would suggest that any transfusion should be preceded by blood typing and cross-match wherever possible. Recent large retrospective surveys have documented a prevalence of only 3% for transfusion reactions in the dog2. The sensitisation of a DEA1 negative dog by an incompatible first transfusion will lead to problems for second and subsequent transfusions. The feline blood group system is less complex3. Cats may be of blood groups A (genotype A/A or A/B), B (genotype B/B) or AB. The A allele is dominant over the B allele and the phenotype AB is the result of a third allele allowing co-dominant expression of both A and B. The AB allele is recessive to the A allele but dominant over the B allele. Blood group B is more prevalent amongst particular purebred cats including the British short hair, Birman and Rex (2045%), Abyssinian, Persian and Somali (1120%). All Siamese, Burmese and Tonkinese cats that have been tested are reported to be blood group A. Cats of

blood group A uncommonly have anti-B alloantibody in their serum, but this is invariably of low titre. Conversely, type B cats usually have high-titred anti-A alloantibody in the circulation, whereas type AB cats have neither alloantibody. Various calculations of the risk of incompatible transfusion can be made on the basis of the known prevalence of feline blood group antigens within a geographical area4, but in general terms the likelihood of incompatible first transfusion in a cat is much greater than for a dog.
Testing for Blood Groups and Cross-Matching

There are a number of reference laboratories in the US where it is possible to submit samples for canine blood typing on a commercial basis, but this is not the case in Europe. Feline blood typing is more readily available, as it does not require the use of specific antisera. In the past few years, a commercially available card system has come on-line that enables typing of cat blood as A, B or AB5. The same manufacturer produces a card system for determination of the DEA1 status of dogs. These cards are simple to use, and provide a rapid and accurate result. Full instructions for use come with these test kits, but in this presentation the methodology will be described. In addition, two further commercially based systems are available which utilise a different, tube-based, technology. These have not been as widely evaluated as the card typing system. The cross-match procedure involves performing both a major cross-match (recipient serum with donor erythrocytes) and minor cross-match (donor serum with recipient erythrocytes) in order to detect the presence of haemagglutinins or haemolysins in the serum 6. A laboratory-based cross match will generally be performed in a microtitre system and the inclusion of fresh complement or antisera might be used in some laboratories. Cross-matching is also readily undertaken in practice, and either a tube-based (preferable) or rapid slide-based methodology can be used. EDTA and clotted (serum) samples will be required from the recipient and any donor animals. In the tube-based test EDTA blood is first washed by thrice repeated centrifugation and suspension of the red cell component in physiological saline, and a 4% red cell suspension is made (0.2 ml packed red cells + 4.8 ml saline). Serum is separated from the clotted sample. Four test tubes are then set up. The first comprises the major cross match and includes one volume of recipient serum and one volume of donor red cell suspension. The second tube is the minor cross-match consisting of one volume of donor serum with one volume of recipient erythrocyte suspension. Two control tubes involve mixing equal volumes of donor serum with donor cell suspension, and recipient serum with recipient erythrocyte suspension. The volumes used in this procedure might range typically from 2 drops to 0.5 ml. The tubes are incubated at 37oC if possible (or at room temperature) for a 30 to 60 minute period. After this time, if the red cells have not pelleted to the base of the tube, the tubes may be lightly centrifuged (1000 rpm, 30 seconds). Tubes are assessed for evidence of haemagglutination or haemolysis indicative of an immunological reaction. If there is any doubt, a small sample of the red cells can be removed, placed on a microscope slide under a coverslip for microscopic examination. The more rapid (but less precise) slide-based test also involves preparation of serum and red cell suspension from recipient and donor animals. In this test, the equal volumes of these reagents (in the same pattern as described above for major and minor cross-match and controls) are added to clean glass microscope slides. The reactants may be mixed with an applicator stick and then the slide gently rocked for a 2 minute period before assessing for haemagglutination. The slides may be cover slipped for microscopic assessment.
SUMMARY

The canine and feline blood group antigen systems differ in their nature and complexity, so whole blood transfusion must be considered separately in each species. Incompatibility in blood group antigens between donor and recipient animals and the presence of spontaneously arising or induced alloantibodies in either recipient (most commonly) or donor (rarely) may lead to blood transfusion reactions. For these reasons, pre-transfusion blood typing and cross-matching is best clinical practice in all cases. Blood typing and cross-matching may be performed by commercial laboratories or in the practice laboratory and this presentation describes the methodology applicable to in-house performance of these tests.

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