Penn Veterinary Medicine | University of Pennsylvania

Blood Typing and Crossmatching

Canine blood types

Blood types are genetically determined markers on the surface of red blood cells. They are specific to each species and are antigenic. A set of blood types of two or more alleles makes up a blood group system. More than a dozen blood group systems have been described in dogs. The current nomenclature is listed as Dog Erythrocyte Antigen (DEA) followed by a number. Red blood cells from a dog can either be positive or negative for any blood group system other than the DEA 1 system. For example, a dog’s red cells can be DEA 3-positive or DEA 3-negative. The DEA 1 system, however, has at least two subtypes: DEA 1.1 (also known as A1) and DEA 1.2 (also known as A2). Thus, a dog’s red cells can be DEA 1.1-positive or -negative, and DEA 1.1-negative cells can be DEA-1.2 positive or negative. Very limited surveys on the frequency of canine blood types have been reported. Some blood types are rare (e.g., DEA 3), whereas others are more common (DEA 4).

Clinically, the most severe antigen-antibody reaction is seen with the DEA 1.1 antigen. Significant naturally occurring alloantibodies are not seen in the dog; therefore, antigen-antibody reactions are not likely to occur on initial transfusion. However, dogs that are DEA 1.1-negative can develop alloantibodies to DEA 1.1 from a mismatched first transfusion. These anti-DEA 1.1 antibodies can develop within a few days from initial transfusion and can potentially destroy the donor’s red blood cells, ultimately minimizing the benefits of the transfusion. However, a previously sensitized DEA 1.1-negative dog can experience an acute hemolytic transfusion reaction following transfusion of DEA 1.1-positive blood. Transfusion reactions may also occur after a previously transfused (and now sensitized) dog receives blood that is mismatched for any red cell antigen other than DEA 1.1. These reactions may occur as early as four days after sensitization. For example, a previously sensitized DEA 4-negative dog experienced an acute hemolytic transfusion reaction while receiving DEA 4-positive blood. In an emergency situation, or with specific medical conditions that preclude conclusive typing (e.g., autoagglutination in an IMHA patient), DEA 1.1-negative blood should be used to avoid sensitization to the DEA 1.1 antigen.

Feline Blood Types

One blood group system, the AB system, has been well-defined in the cat. It contains three blood types: A, B and the extremely rare AB. Nearly all domestic short hair (DSH) and domestic long hair (DLH) cats in the United States have type A blood. Many purebred cats (and some DSHs) have been identified with type B blood. The proportion of type A and B varies not only among the different breeds, but also geographically. The rare type AB blood has both the A and B antigen on the red cell surface. Many other blood group systems are thought to exist (e.g., Mik), but have not yet been fully characterized.

Cats differ from dogs in that they have significant naturally-occurring alloantibodies against the other blood group. Cats with type B blood have very strong naturally-occurring anti-A alloantibodies, whereas type A cats have relatively weak anti-B alloantibodies. When administering type B blood to a type A cat, there may not be any obvious clinical reaction, but the transfused red cells have a half life of approximately 2 days. Ultimately, this has no positive effect on the patient. In administering type A blood to a type B cat, the red cell survival can be minutes to hours with severe clinical signs, sometimes fatal. Administration of a small amount of blood to test for incompatibility is not an acceptable procedure. Life-threatening acute hemolytic transfusion reactions can be observed with administration of as little as 1 milliliter of AB-incompatible blood. These reactions can be avoided by typing donors and patients. If blood typing is not available, a blood crossmatch should be performed to ensure blood compatibility. The extremely rare blood type AB cat lacks anti-A and anti-B alloantibodies and can be safely transfused with type A packed red blood cells if type AB blood is not available.

Blood crossmatch

A blood crossmatch (BCM) is performed to detect serological incompatibility by identifying antibodies in donor or recipient plasma against recipient or donor red blood cells. A BCM is divided into two parts: the major crossmatch consists of mixing the patient’s plasma with the donor’s red blood cells; the minor crossmatch consists of mixing the donor’s plasma with the patient’s red blood cells. Of the two tests, the major blood crossmatch is much more important in determining survival of the transfused red blood cells.

Dogs lack significant naturally occurring alloantibodies; therefore, they may be safely transfused without a BCM prior to the first transfusion. However, all dogs that have received RBC transfusions more than four days previously must be crossmatched before receiving any additional RBC transfusions. Since cats have naturally occurring alloantibodies and may experience a severe reaction to their first transfusion, a BCM should be performed prior to any blood transfusion if blood typing is not available. A BCM is typically not necessary for a first transfusion if the blood types of the feline recipient and donor are known. As with dogs, feline patients that have received RBC transfusions more than four days previously should be crossmatched before receiving any additional RBC transfusions.

Procedure for BCM

1. Collect blood into an EDTA tube from recipient and potential donor(s).
2. Centrifuge (1000 x g for 5 min) to separate plasma from RBCs, remove plasma from each sample with a pipette and transfer plasma to clean, labeled glass or plastic tubes. Note any hemolysis.
3. Wash RBCs 3 times with PBS:
   1. add 4–5 ml of PBS,
   2. mix well,
   3. centrifuge 1–2 minutes,
   4. remove saline, leaving pellet of RBCs at bottom of tube.
4. Resuspend RBC pellet with PBS to make a 3–5% RBC suspension.
5. Prepare for each donor 3 tubes labeled major, minor, and recipient control. Add to each tube 2 drops (50 µl) of plasma and 1 drop (25 µl) of RBC suspension as follows:
   1. major recipient plasma + donor RBCs,
   2. minor donor plasma + recipient RBCs,
   3. recipient control recipient plasma + recipient RBCs.
6. Mix gently and incubate for 15–20 minutes at 37° C in a warm water bath.
7. Centrifuge for 15 seconds at 1000 x g.
8. Examine supernatant for hemolysis.
9. Gently resuspend the button of RBCs by tapping tube and examine for macroscopic agglutination ? classify as 1+ (fine), 2+ (small), 3+ (large), or 4+ (one large agglutinate).

An autocontrol sample of recipient RBCs and plasma is included because some recipients may have autoagglutination interfering with the BCM. If the recipient control is positive (i.e., agglutination is present), one cannot draw conclusions about blood compatibility between patient and donors. Any hemolysis and/or agglutination in the major or minor BCM (but not the control) indicate an incompatibility and the need to choose a new donor. The minor BCM should be compatible in dogs since canine donor plasma should not contain significant antibodies. Feline patients must be given type-specific plasma products due to the presence of naturally occurring alloantibodies.

A compatible BCM does not prevent sensitization or delayed transfusion reactions; it simply indicates that at the present time no detectable antibodies against the RBCs exist.

For further information, please visit the Transfusion Medicine Laboratory.