Common Immunologic Laboratory Tests and Blood Groups in Veterinary Medicine

Evaluation of Immunologic Laboratory Tests

• General Purpose of Immunologic Tests

  • Tests are designed to detect specific infectious agents in animals.

  • Many of these diagnostic tools are commercially available in convenient kit forms.

• Sensitivity

  • Defined as the ability of a test to correctly identify all animals that are truly positive for a given reaction procedure.

• Specificity

  • Measures the number of false positives produced with a given reaction procedure.

• Reliability Limits

  • In clinical practice, no immunologic test possesses both $100\%$ sensitivity and $100\%$ specificity.

Sample Collection and Handling Protocols

• Specimen Requirements

  • Nearly all serologic tests mandate the use of either serum or plasma.

  • Whole blood is generally not suitable for shipment to diagnostic laboratories.

• Collection Tools and Identification

  • Vacutainer tubes are the preferred method for collection.

  • Red top tubes are utilized when serum is required.

  • Lavender top tubes are utilized when plasma is required, unless specific instructions state otherwise.

  • Adhesion to strict requirements for reference laboratories is essential; directions must be read carefully and followed precisely.

• Serum Processing Steps

  • Allow the blood to clot for $20$ to $30\,minutes$ at room temperature.

  • Centrifuge the sample for $10\,minutes$ at a speed $≤ 1500\,rpm$.

• Plasma Processing Steps

  • Centrifuge the sample immediately following collection.

• Post-Processing Storage

  • Pipette the resulting serum or plasma into a transfer tube and label appropriately.

  • The sample should be frozen or refrigerated for later use.

Methodologies for Testing Humoral Immunity

• Enzyme-Linked Immunosorbent Assay (ELISA)

  • This is the most frequently used immunologic test in veterinary medicine.

  • It serves as an accurate method for detecting specific antigens, including viruses, bacteria, parasites, and hormones.

  • It is also used to test for antibodies in serum.

  • Common applications include testing for Heartworm, Feline Leukemia (FeLV), Feline Immunodeficiency Virus (FIV), Parvovirus, and Progesterone levels.

  • Mechanism of Action:

    • A monoclonal antibody is bound to the walls of a well, a test tray, or a plastic wand.

    • If the specific antigen is present in the sample, it binds to the fixed antibody.

    • A second enzyme-labeled antibody is added, which also binds to the antigen.

    • The system undergoes a rinsing phase to remove unbound materials.

    • Color-producing substrates are added, which react with the enzyme to indicate the presence of the antigen.

• Competitive Enzyme-Linked Immunosorbent Assay (CELISA)

  • This variation utilizes patient antigens along with enzyme-labeled antigens and monoclonal antibodies.

  • The intensity of the color produced varies according to the concentration of the antigen.

  • It is specifically used for detecting Equine Infectious Anemia.

• Latex Agglutination

  • Uses small, spherical latex particles coated with an antigen that are suspended in water.

  • If serum containing the matching antibody is added to the suspension, visible agglutination (clumping) occurs.

  • This method is used to detect Bovine Brucellosis.

• Rapid Immunomigration (RIM)

  • Also known as immunochromatography or lateral flow immunoassay.

  • Components include colloidal gold, enzymes, and color reagents or units of agglutinated latex particles.

  • The test cassette membrane contains antibodies where the sample is applied.

  • A positive result is indicated by two distinct areas of color: one for the test result and one for the control.

Immunology Analyzers

• In-Practice vs. Reference Units

  • Immunology analyzers are available for clinical practices but are not currently common.

  • Practice-level units typically only read the test results.

  • Larger units found in reference laboratories have the capacity to read multiple tests simultaneously.

Blood Group Antigens and Alloantibodies

• Surface Antigens

  • Red Blood Cell (RBC) antigens are structures on the cell surface that react with antibodies found in the plasma of other animals.

  • These specific surface markers are genetically determined.

  • Antigen-antibody reactions during blood transfusions can result in the clumping or lysis of RBCs.

• Alloantibodies

  • These are naturally occurring antibodies found in cats, cattle, sheep, and pigs.

  • Crossmatching is necessary for breeding females to avoid the potential production of antibodies against neonate RBCs.

Veterinary Blood Banks

• Availability and Components

  • Blood banks have increased the availability of components such as packed Red Blood Cells (pRBCs) and platelet-rich plasma.

  • This availability has significantly improved emergency and critical care capabilities.

  • Veterinary blood banks perform typing and crossmatching to ensure patient safety during component transfusions.

Canine Blood Groups and Transfusion Dynamics

• Nomenclature and Variety

  • Dogs possess more than a dozen blood groups.

  • The standard nomenclature used is Dog Erythrocyte Antigen (DEA).

  • JUST FOCUS ON DEA 1.1 + or -

• Major Canine Blood Groups

  • DEA 1: Includes subgroups $DEA\,1.1+$ , $DEA\,1.2+$, $DEA\,1.3+$, $DEA\,1.1-$, $DEA\,1.2-$, and $DEA\,1.3-$.

  • DEA 3

  • DEA 4

  • DEA 5

  • DEA 7

• Clinical Significance of DEA 1.1

  • DEA 1.1 is the most clinically significant because it elicits the greatest antigen response and is responsible for many transfusion complications.

  • Approximately $50\%$ of all dogs are positive for DEA 1.1.

  • Naturally occurring DEA 1.1 antibodies are not known to exist.

  • Transfusion Reactions:

    • The first transfusion of $DEA\,1.1+$ blood into a $DEA\,1.1-$ dog may not cause an immediate reaction.

    • However, the recipient can develop antibodies, potentially causing delayed problems up to a week later.

    • If a $DEA\,1.1-$ dog that has been sensitized (previously received $DEA\,1.1+$ blood) is given $DEA\,1.1+$ blood again, a severe reaction can occur in less than one hour.

Feline Blood Groups and Related Conditions

• AB System

  • Cats utilize the AB system, consisting of types A, B, and AB.

  • Type A blood is the most common in the United States, resulting in a low incidence of transfusion reactions for these cats.

  • Type B blood is found in specific purebreds and certain geographical regions, such as Australia.

  • Type AB blood is rare.

• Naturally Occurring Feline Antibodies

  • Cats have naturally occurring antibodies against the RBC antigens they lack.

  • Type B cats possess strong anti-A antibodies.

  • Type A cats possess weak anti-B antibodies.

  • Transfusing a type B cat with Type A blood can result in a serious reaction or death.

  • Typed blood and crossmatching are essential for purebred cats.

• Neonatal Isoerythrolysis

  • This condition has been documented in type A and type AB kittens born to type B queens.

Blood Groups in Large Animals

• Cattle, Sheep, and Goats

  • These species possess multiple blood types.

• Horses

  • Horses have more than $30$ blood types categorized into $8$ major groups: A, C, D, K, P, Q, T, and U.

  • Naturally occurring antibodies may exist, or they may develop following vaccinations containing equine tissue/transplacental immunization.

  • Reactions to transfusions in horses are commonly fatal.

  • Mare-Foal Incompatibility Test:

    • A crossmatch used to detect antibodies in the mare's serum or colostrum against the foal's RBCs to prevent neonatal isoerythrolysis.

    • Crossmatching is mandatory before any equine transfusion.

Laboratory Methods for Blood Typing and Crossmatching

• The Tube Method

  • Requires the use of antisera (antibodies specific for each blood type of a species).

  • Requires whole blood collected in EDTA, heparin, or acid-citrate-dextrose anticoagulant.

  • The process involves several steps and requires both macroscopic and microscopic examination of each tube.

  • This method is not practical for large animal blood typing because it would require thousands of different antisera.

• The Card Agglutination Test

  • Available as Rapid Vet H canine and feline test kits.

  • It is considered a rapid and accurate method for blood typing.

• Immunochromatographic Assay

  • Two test kits apply this principle instead of using agglutination.

  • A control band is used to detect separate antigens on the RBCs.

• Crossmatching Procedures

  • Crossmatching is performed when antisera for typing is unavailable.

  • It requires both serum and whole blood samples from the donor and recipient.

  • Major Crossmatch: A few drops of serum from the recipient are added to a few drops of washed pRBCs from the donor.

  • Minor Crossmatch: A few drops of serum from the donor are mixed with washed pRBCs from the recipient.

  • Commercial kits are available for performing these crossmatches.

Summary of Clinical Essentials

• Critically ill patients require blood typing and both a major and minor crossmatch before any transfusion.

• Species vary in the number of blood type groups they possess.

• The majority of domestic cats have type A blood.

• Typing and crossmatching are the primary methods to minimize potential transfusion reactions.