Immunologic Laboratory Tests and Blood Groups in Veterinary Medicine

Evaluation of Immunologic Tests

• To accurately detect specific infectious agents, various immunologic tests are employed, many of which are commercially available in kit form.

• These tests are evaluated based on two primary metrics: sensitivity and specificity.

• Sensitivity refers to 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.

• It is a standard principle that no immunologic test possesses both $100\%$ sensitivity and $100\%$ specificity.

Sample Collection for Serology

• The vast majority of serologic tests require either serum or plasma for analysis.

• Diagnostic laboratories generally indicate that whole blood should not be sent for testing.

• A Vacutainer is the preferred collection vessel:

  • A Red top tube is utilized if serum is required.

  • A Lavender top tube is utilized for plasma, unless specific instructions note otherwise.

• Reference laboratories often have strict requirements; directions must be read carefully and followed exactly to ensure sample integrity.

Handling Serologic Samples

• Serum Processing:

  • Blood should be allowed to clot for $20$ to $30$ minutes at room temperature.

  • The sample is then centrifuged for $10$ minutes at a speed of $\le 1500\,\text{rpm}$.

• Plasma Processing:

  • Centrifugation should occur immediately after the collection of the sample.

• Once processed, the serum or plasma should be pipetted into a transfer tube and properly labeled.

• Samples can be frozen or refrigerated for later use.

Enzyme-Linked Immunosorbent Assay (ELISA)

• The ELISA is the most commonly used immunologic test in veterinary practice.

• It serves as an accurate method for detecting specific antigens, including:

  • Viruses

  • Bacteria

  • Parasites

  • Hormones

• In addition to antigens, it can test for antibodies in serum.

• Test Kit Contents: Specific antigens are contained within the kit for detecting various conditions such as heartworm, feline leukemia (FeLV), feline immunodeficiency virus (FIV), parvovirus, and progesterone levels.

• Mechanism of Action:

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

  • If the target antigen is present in the sample, it binds to this antibody.

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

  • The system is rinsed to remove unbound materials.

  • Color-producing substrates are added to the system.

  • These substrates react with the enzyme to produce a color change, indicating the presence of the antigen.

Competitive Enzyme-Linked Immunosorbent Assay (CELISA)

• This variation of the ELISA uses the patient's antigen.

• It involves the use of enzyme-labeled antigens as well as monoclonal antibodies.

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

• It is specifically used for testing for Equine Infectious Anemia (EIA).

Latex Agglutination and Rapid Immunomigration

• Latex Agglutination:

  • This test utilizes small, spherical latex particles that are coated with a specific antigen and suspended in water.

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

  • This method is used to test for Bovine Brucellosis.

• Rapid Immunomigration (RIM):

  • Also known as lateral flow immunoassay.

  • This method utilizes colloidal gold, enzymes, and color reagents or agglutinated latex particles.

  • Antibodies are present within the membrane of the test cassette where the sample is applied.

  • A positive result is indicated by the appearance of color in two distinct areas: the test area and the control area.

Immunology Analyzers

• While available for use within individual veterinary practices, immunology analyzers are not common in that setting.

• In-practice units typically only read the results of tests.

• Larger units are utilized in reference laboratories, which are capable of reading multiple tests simultaneously.

Blood Group Antigens

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

• These specific surface markers are determined genetically.

• Antigen-antibody reactions can occur during blood transfusions, which may result in the clumping (agglutination) or lysis (destruction) of RBCs.

• Alloantibodies:

  • These are naturally occurring antibodies.

  • They are found in cats, cattle, sheep, and pigs.

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

  • For dogs you only get one free transfusion ; subsequent transfusions require careful crossmatching to prevent serious transfusion reactions due to alloantibodies that may develop.

Veterinary Blood Banks

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

• This availability has led to improvements in emergency and critical care settings.

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

Canine Blood Groups and Transfusions

• Dogs have more than a dozen blood groups.

• DEA 1.1(+/-) is the most used in dogs

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

• Major blood groups include:

  • DEA 1: This group is further divided into subgroups including $DEA\,1.1^{+}$, $DEA\,1.2^{+}$, $DEA\,1.3^{+}$, $DEA\,1.1^{-}$, $DEA\,1.2^{-}$, and $DEA\,1.3^{-}$.

  • DEA 3

  • DEA 4

  • DEA 5

  • DEA 7

• DEA 1.1:

  • This is the most clinically significant type.

  • It elicits the greatest antigen response and is responsible for the majority of transfusion complications.

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

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

• Transfusion Reactions:

  • A first-time transfusion of $DEA\,1.1^{+}$ blood into a $DEA\,1.1^{-}$ dog may not result in an immediate reaction; however, the dog can develop antibodies against it.

  • This sensitization can cause problems up to a week later.

  • If a sensitized $DEA\,1.1^{-}$ dog (having 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 Types and Complications

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

• Very few cats possess the AB blood type.

• The majority of cats in the United States have type A blood, which is associated with a low incidence of transfusion reactions.

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

• Cats have naturally occurring antibodies (alloantibodies) to the RBC antigens they lack.

• Compatibility:

  • 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 lead to a serious reaction or death.

  • Consequently, purebred cats require rigorous typing and crossmatching.

• Neonatal Isoerythrolysis:

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

Blood Types in Large Animals

• Cattle, Sheep, and Goats: These species are characterized by having multiple blood types.

• Horses:

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

  • Naturally occurring antibodies may exist, but they are more commonly present after a vaccination containing equine tissue or through transplacental immunization.

  • Crossmatching is mandatory before any transfusion because horse transfusion reactions are commonly fatal.

  • The mare-foal incompatibility test involves crossmatching the mare's serum (or colostrum) with the foal’s RBCs to prevent neonatal isoerythrolysis.

Blood Typing Methodologies

• The Tube Method:

  • Requires specific antisera, which are antibodies for each possible blood type of a species.

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

  • This involves several steps, and each tube must be examined both macroscopically and microscopically.

  • This method is not practical for large animal blood typing because thousands of antisera would be required.

• The Card Agglutination Test:

  • Commercially available as Rapid Vet H canine and feline test kits.

  • Provides a rapid and accurate blood typing result.

• Immunochromatographic Assay:

  • Some kits use this principle instead of agglutination.

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

Crossmatching Procedures

• Crossmatching is performed if antisera are unavailable for blood typing.

• Both major and minor crossmatches require serum and whole blood samples.

• 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 these procedures.

Summary of Clinical Considerations

• Critically ill patients must undergo blood typing and crossmatching prior to receiving a transfusion.

• Different animal species have varying numbers of blood type groups.

• While the majority of cats have type A blood, typing and crossmatching are essential to minimize potential reactions.

• Safe transfusion practice require both a major and a minor crossmatch.