Ensuring a safe blood supply: Part II (Proceedings)

Bleeding animals often present to the emergency room in advanced stages of disease, on the verge of cardiovascular collapse, and in need of immediate therapeutic intervention. Although a definitive diagnosis is important in the ultimate treatment of these patients, stabilizing the patient's emergent clinical problems is critical. This may include controlling hemorrhage and replacing lost blood volume with the appropriate intravenous fluid solutions and/or blood components, improving oxygen-carrying capacity with oxygen and red blood cell support, and taking all necessary measures to combat shock. If oxygen-carrying support or hemorrhage control is indicated, the benefit of transfusion therapy must be weighed against its inherent risks.

Pretransfusion testing is necessary to ensure the best possible results of a blood transfusion. It includes testing of the donor, selection of appropriate donor units based on the patient's blood type, and blood crossmatching. Although pretransfusion testing will help to determine pre-existing incompatibility between the donor and recipient, normal survival of transfused cells in the patient's circulation cannot be guaranteed. Blood samples for initial testing should always be collected from patients prior to infusion of any donor blood products.

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 4 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.

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 DSH/DLH) 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 given that 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.

Canine and feline blood typing methods

Various methods have been developed for blood typing cats and dogs in both the clinical laboratory and practice setting. The principle of blood typing methods is the presence of a hemagglutination reaction that results when a RBC surface antigen binds with known polyclonal or monoclonal antibodies (within seconds to minutes).

Clinical laboratory blood typing methods are slightly more complex and/or require specialized equipment. They include the following: tube and slide method using a polyclonal antibody and agglutination reagent (wheat germ lectin, Triticum Vulgaris) in the cat, tube assay using polyclonal antisera reagents in the dog, and gel column diffusion technology using monoclonal antibodies in the dog and cat.

Currently, there are two commercially available point-of-care assays for canine and feline blood typing: a card-based agglutination test and an immunochromatographic assay. Both of these tests are user friendly and yield reasonably accurate results, making them invaluable in the emergency setting. With both canine and feline blood typing, blood samples must first be evaluated for autoagglutination. If macroscopic autoagglutination is present, washing red blood cells three times with phosphate buffered saline (PBS) (see blood crossmatch procedure below) may eliminate the problem, otherwise blood typing cannot be performed.

Card-Based Agglutination Test

RapidVet™-H (Canine DEA 1.1)

This blood typing test card is intended for use in classifying dogs as DEA 1.1 positive or negative. The assay is based on the agglutination reaction that occurs when erythrocytes which contain DEA 1.1 antigen on their surface membranes interact with a monoclonal antibody specific to DEA 1.1. Each card has 3 visually-defined wells identified as "DEA 1.1 Positive Control", "DEA 1.1 Negative Control", and "Patient Test". One drop of whole blood (in EDTA) and one drop of phosphate buffered saline (PBS) are mixed onto lyophilized reagents within each well, being careful to avoid cross contamination between the wells. In the "Patient Test" well, the monoclonal antibody is reconstituted to form an antiserum and then mixed with whole blood from the patient. DEA 1.1 positive erythrocytes react with the antiserum causing agglutination. If agglutination is present in the "Patient Test" well, it is then graded from 0-4+. The antiserum is completely nonreactive with DEA 1.1 negative erythrocytes. "Auto-Agglutination Saline Screen" cards are supplied separately if autoagglutination is suspected.

RapidVet™-H (Feline)

A similar blood typing test card is available to classify cats as type A, B, or AB. The assay is based on the agglutination reaction that occurs when erythrocytes interact with a monoclonal antibody specific for the A antigen and/or an anti-B solution (wheat germ lectin, Triticum vulgaris, which causes agglutination of type B cells). Erythrocytes from type A cats will agglutinate with anti-A monoclonal antibodies (well labeled A on card) and erythrocytes from type B cats will agglutinate with anti-B solution (well labeled B on card). Erythrocytes from type AB cats will agglutinate with both anti-A and anti-B reagents. The third well on the card serves as the autoagglutination saline screen and must be negative in order to interpret results.

*The RapidVet™-H (Canine DEA 1.1 and Feline) blood typing test cards are manufactured by DMS Laboratories, Flemington, NJ 08822, 800-567-4367, www.rapidvet.com.

Immuno-Chromatography Assay

DME VET Canine DEA 1.1 Blood Typing or Feline AB Blood Typing

An alternative method of DEA 1.1 blood typing for dogs and AB typing for cats has recently become available for use in practice. The technique uses immuno-chromatography, similar to many SNAP ELISA tests, rather than agglutination. The control band detects a separate antigen on the red blood cells. Autoagglutination does not seem to interfere with this technique.

The canine test uses a monoclonal anti-DEA 1.1 antibody strip impregnated onto a paper strip and a second control antibody to a universal RBC antigen as a control. A 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 cells have successfully diffused up the length of the strip.

The feline test works in the same way; however, there is an area containing an anti-A monoclonal antibody, an area containing an anti-B monoclonal antibody, and a control antibody against a common feline RBC antigen, allowing determination of blood type A, B or AB.

*DME VET cartridges are manufactured by Alvedia, Lyon, France, http://www.alvedia.com.

Blood Crossmatch

Dogs lack significant naturally occurring alloantibodies; therefore, they may be safely transfused without a blood crossmatch (BCM) prior to the first transfusion. However, all dogs that have received RBC transfusions more than 4 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 4 days previously should be crossmatched before receiving any additional RBC transfusions.

A BCM is performed to detect serological incompatibility by identifying antibodies in donor or recipient plasma against recipient or donor red blood cells. The test is composed of three individual tests:

• Recipient control → recipient plasma + recipient RBC

• Major blood crossmatch → recipient plasma + donor RBC

• Minor blood crossmatch → donor plasma + recipient RBC

An autocontrol sample of recipient RBC 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, delayed transfusion reactions or non-hemolytic transfusion reactions; it simply indicates that at the present time there are no detectable antibodies against the RBC. Keep in mind that sensitization may develop later from the transfusion of any RBC membrane antigen not produced by the patient. Additionally, there are many types of transfusion reactions not caused by RBC membrane antigen incompatibilities (e.g., febrile non-hemolytic transfusion reactions, transfusion of damaged/lysed blood).

Blood Crossmatch – Tube Method

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 RBC. Remove plasma from each sample with a pipette, and transfer plasma to clean, labeled glass or plastic tubes. Note any hemolysis.

3. Wash RBC pellet three times with phosphate buffered saline (PBS):

a. add 4-5 ml of PBS

b. mix well

c. centrifuge 1-2 minutes

d. remove saline, leaving pellet of RBC 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:

• Recipient control → recipient plasma + recipient RBC (perform once)

• Major blood crossmatch → recipient plasma + donor RBC

• Minor blood crossmatch → donor plasma + recipient RBC

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 RBC by tapping tube and examine for macroscopic agglutination. Classify as 1+ (fine), 2+ (small), 3+ (large), or 4+ (one large agglutinate).

10. If macroscopic agglutination is not observed, transfer a small amount onto a glass slide and examine for microscopic agglutination (microscopic agglutination is of questionable importance). Differentiate between agglutination and rouleaux formation.

RapidVet-H Major and Minor Crossmatch Kit

A commercial kit has recently become available for performing a major and minor BCM on feline and canine blood. The kit contains all of the pipettes and tubes necessary to perform the BCM, requiring nothing more than donor and recipient blood samples and a centrifuge. The kit is not recommended for severely hemolyzed samples or in patients that have recently received Oxyglobin. Similar to other commercial laboratory techniques, the test relies on the fact that agglutinated RBC will not sediment through a viscous gel as effectively as free individual RBC.

References available upon request