BRIEF OVERVIEW

The long-recognized feline AB blood system displays two major blood types, A and B, as well as the rare type AB. The A and the B types are alternate forms of the same gene, alleles; the allele A, for blood type A, is dominant to allele b, which controls blood type B. Although several studies have examined the rare AB type, which co-expresses both A and B antigens phenotypically, its heritability still needs to be elucidated.^3–6 The AB blood group has been found exclusively in breeds in which the B blood type occurs.⁴

Ontogenetically, erythrocyte antigens are developed in utero, whereas antibodies are developed postnatally. Both the cat anti-A and anti-B antibodies are naturally occurring antibodies. The anti-A antibodies are developed earlier than the anti-B antibodies, probably because epitopes similar to the A antigen are more common in the environment or are stronger immunological stimulants than the B antigens.⁷

The feline blood types are clinically important in two circumstances. The possession of a high titer of anti-A antibodies in a B-type cat leads to severe transfusion reactions if receiving blood from an A-type cat. Further, neonatal isoerythrolysis (NI) can occur, especially when a B-type queen nurses A-type kittens, because the antibodies pass through the colostrum.¹

SEROLOGICAL TECHNIQUES FOR FELINE BLOOD TYPE DETECTION

Blood typing and cross-matching are critical components of pretransfusion testing in feline patients, and both techniques help to identify compatible blood donors correctly.⁸ Blood typing identifies blood group antigens on the surface of erythrocytes, whereas cross-matching detects the presence of significant levels of antibodies directed against erythrocyte antigens. However, because blood typing is unable to detect blood group incompatibilities in the absence of naturally occurring alloantibodies, cross-matching remains the only method available to ensure that AB-system–compatible blood is transfused.⁹

Cross-matching refers to the testing that is performed to determine the compatibility of a donated unit of blood with its intended recipient. In short, erythrocytes from the donor unit are tested against the plasma of the patient in need of the blood transfusion. If the patient’s plasma contains antibodies against the antigens present on the donor erythrocytes, agglutination will occur. Agglutination is considered a positive reaction, indicating that the donor unit is incompatible for that specific patient. If no agglutination occurs, the unit is deemed compatible and is safe to transfuse.

The principle of serological blood-typing reactions is based on the identification of macroscopically visible agglutination of erythrocytes following incubation with known antibodies or special agglutinin reagents.¹⁰ Agglutination of erythrocytes signifies a positive reaction, whereas if no agglutination reaction occurs, the test is considered negative for the blood group antigen being examined.

Agglutination reactions were performed initially in test tubes or micro-well plates and were given a grade based on the degree of macroscopic erythrocyte agglutination observed. Grading of agglutination reactions gives an indication of the relative amount of antigen or antibody present. In grading agglutination reactions, a 4+ reaction yields a solid clump of erythrocytes and a 3+ reaction yields several small clumps. A 2+ reaction yields small- to medium-sized clumps with a clear background. A 1+ reaction yields several small clumps with a cloudy background, and a weak+ reaction reveals many tiny aggregates with a cloudy background.

Currently, all commercial serological blood typing tests for cats are based on agglutination reactions. Although the use of agglutination techniques for blood typing remains a simple, sensitive, and reliable test, agglutination techniques are only as sensitive as the antibodies or agglutinins used, so that new blood group antigens may be overlooked without the availability of specific antibodies and/or agglutinins.

Confirmation of blood typing results may be accomplished through the use of a back-typing test in which the plasma or serum of the cat being blood typed is evaluated for alloantibodies. Back-typing involves incubating known type-A and type-B erythrocytes with the patient’s plasma. Plasma from type-A cats typically will agglutinate type-B erythrocytes weakly to moderately. Plasma from type-B cats will agglutinate type-A erythrocytes strongly. The back-typing test is similar to the cross-match test in circumstances in which the blood type of the blood donor is known and assists in confirming the patient’s (transfusion recipient’s) blood type.

The reagents used for feline blood typing are either polyclonal or monoclonal antibodies or lectins. The polyclonal anti-sera created from the serum of type-A and type-B positive cats has been used to blood type cats for decades; in particular, the anti-A serum from type-B cats has high erythrocyte agglutination activity.⁹ The lectin from Triticum vulgaris has replaced the feline anti-B serum because it agglutinates type-B positive erythrocytes preferentially, whereas it agglutinates type-A positive erythrocytes only at higher concentrations.¹¹

Current serological methods used for feline blood typing include the Penn tube or slide agglutination assays (Transfusion Laboratory, Section of Medical Genetics, School of Veterinary Medicine, University of Pennsylvania, Philadelphia), blood typing card tests, and gel column tests. Both the Penn slide and tube assays use anti-A polyclonal serum and T. vulgaris lectin.¹² The Penn tube assay is reliable and has been considered the gold standard and has been utilized as the reference standard in several blood type frequency surveys.¹⁰

A tube assay using monoclonal antibodies to identify both A and B blood group antigens has been developed (Shigeta Animal Pharmaceuticals Inc, Komoridani, Oyabe City, Toyama Pref, Japan), thereby eliminating polyclonal antibodies and lectins entirely.¹³ The Penn slide assay is a simplified version of the tube assays and allows typing of whole blood, thus lending itself to use in a practice setting. The tube methods have been confined mostly to diagnostic laboratories and research institutions, given their greater complexity and the technical expertise required to perform the assay. The card test (DMS RapidVet-H [feline] blood typing card test, DMS Laboratories Inc, Flemington, NJ) uses a monoclonal anti-A antibody for the identification of type-A erythrocytes and the T. vulgaris lectin for identification of type-B erythrocytes. Use of a gel matrix column to fix the blood typing agglutination reaction has been developed for use in cats (DiaMed ID-Card Anti A+B [Cat], DiaMed AG, Cressier sur Morat, Switzerland). Like the other methods of determining blood type in cats, the gel matrix column uses an anti-A antibody and T. vulgaris lectin.

In a comparison of the previously mentioned blood typing assays, using the Penn tube test as a reference standard, Giger and colleagues found that the blood typing card tests allow identification of both type-A and type-B cats; however, weak reactions of type-AB blood with the monoclonal type-A antibody were a concern. Therefore type-AB and type-B results should be confirmed either by laboratory alloantibody testing or using another technique.¹⁰ They further concluded that the gel test and the Japanese tube test appear to be reliable clinical laboratory methods for feline blood typing.

THE Mik ANTIGEN: A NEW BLOOD TYPE

The feline A, B, and AB blood group system is the only internationally recognized blood group system in cats. However, blood group antigen incompatibilities unrelated to the AB blood group system have been suspected after rare incompatible cross-match results from AB-type–compatible donors and recipients and because of very rare hemolytic transfusion reactions after AB-type–compatible blood transfusion.

In 2005, Weinstein and colleagues,¹⁴ using standard tube and novel gel column cross-matching techniques, identified the presence of a clinically relevant alloantibody, formed against a newly discovered feline erythrocyte antigen, which was named Mik. The incidence of the Mik antigen in the feline population is not known at this time; however, the clinical significance of the Mik antigen was established after an acute hemolytic transfusion reaction from an inadvertent transfusion of Mik-positive blood to a Mik-negative renal transplant patient at the Veterinary Hospital of the University of Pennsylvania. Identification of previously unknown blood types typically follows the investigation of an unexpected acute hemolytic transfusion reaction.

Routine screening of potential blood donors and transfusion recipients for the Mik antigen may become necessary as part of pretransfusion blood typing pending the outcome of prevalence studies. Until such time, these transfusion reactions highlight the need to perform a cross-match before blood transfusion in the feline patient, even when there have not been any previous transfusions.

MOLECULAR STRUCTURES OF FELINE BLOOD TYPES

The earliest studies investigating glycoconjugates on feline erythrocyte membranes revealed the presence of seven different gangliosides. These first studies were carried out on cats of unknown blood types.^15,16 Later, the molecular structures of the antigens determining the blood type of the AB system were defined by Butler et al in 1991.¹⁷

Thin-layer chromatography suggested that the antigens are defined principally by the form of the neuraminic acid residues present on a ceramide dihexose backbone on the surface of the erythrocytes.¹⁷ The major glycolipid and antigen of type-A cats is the disialoganglioside NeuGc-NeuGc-Galactose-Glucose-Ceramide ([NeuGc]₂G_D3), in which NeuGc represents N-glycolyl neuraminic acid. The major glycolipid of type-B cats is the disialoganglioside NeuAc-NeuAc-Galactose-Glucose-Ceramide ([NeuAc]₂G_D3), with NeuAc representing N-acetylneuraminic acid.¹⁷

Further studies on the composition of the erythrocyte antigens showed that the A-type cat also contains a minor amount of (NeuAc)₂G_D3 and two intermediate forms such as NeuAc-NeuGc-G_D3 and NeuGc-NeuAc-G_D3.^12,18 Furthermore, minor amounts of monosialogangliosides such as (NeuAc)G_M3 and (NeuGc)G_M3, the latter not being found in type-B cats, were discovered (Table 61-1). Additionally, trisialoganglioside seems to be present in all of the three blood types.¹²

Table 61-1 Composition of the Gangliosides of Feline Erythrocyte Membrane Determining the Three Blood Types

Cats of the rare AB type have both of the sialic acids, NeuGc and NeuAc, co-expressed on the erythrocyte membrane; even though they have the same composition as type-A cats, the relative amounts differ from those in type-A cats.¹⁸ In contrast with previous findings that suggested a minor amount of B antigen in the AB-type than in the B-type,^4,12,18 a recent study demonstrated a higher expression in type-AB cats of (NeuAc)G_D3 as compared to (NeuGc)G_D3.⁵ The amount of (NeuAc)G_D3 is reported to be similar to that in type-B cats, whereas the antigen (NeuGc)G_D3 was confirmed to be less expressed in type-AB than in type-A cats.⁵

Surprisingly, this study, which included four AB cats and used several types of monoclonal antibodies, showed differences between the A antigen of type-A and type-AB cats.⁵ The authors concluded that at least two different phenotypes exist within the AB blood type, varying in amount and form of the A antigen. Therefore several slightly varying potential antigens may be present on the feline erythrocytes, suggesting that cats may have additional “subtypes.” Extensive studies will be required to shed light on this hypothesis.

NeuAc and NeuGc are two of the most abundant forms of sialic acids in mammalian cells, with NeuGc being the predominant type. NeuGc is produced from NeuAc by a pathway involving the enzyme cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMP-NeuAc).¹⁹ CMP-NeuAc hydroxylase is an enzyme that converts NeuAc to NeuGc, which is expressed in all mammals except human beings. The absence of NeuGc in human beings is due to a partial deletion in the gene (CMAH) encoding the enzyme.^20,21 In human beings, NeuGc is an oncofetal antigen expressed in certain tumors but not in the normal adult tissue.²² However, more recent studies found the presence of NeuGc not only in several human carcinomas but also in some healthy human tissues, raising the question as to whether other pathways could generate this molecule because the CMAH gene is nonfunctional. Bardor and colleagues found that exogenous free NeuGc can be incorporated via pinocytic/endocytic pathways, delivered to the lysosome, and then reach the cytosol by a sialic acid transporter.²³ Red meat and milk products can be dietary sources of free NeuGc.²³ Whether NeuGc is generated by alternate pathways in cats or if it is absorbed from dietary sources has not been studied to date.