Antiglobulin (Coombs') tests are blood tests that identify the causes of immune-mediated anemia or hemolysis. Antiglobulin tests utilize an antibody to human immunoglobulin in order to detect antibody coated (sensitized) cells. In addition to being a medical test that is ordered by a physician, the antiglobulin test is a procedure employed in various blood banking and immunology methods in order to detect immune complex formation. This test is also the basis for some blood typing tests.
Antiglobulin tests are used to detect antibodies in serum or attached to cells. The test is ordered as a medical laboratory test to aid in the differential diagnosis of anemia. Anemia refers to blood with abnormally low oxygen-carrying capacity. The hemoglobin in red blood cells carries oxygen. Anemia may be caused by low numbers of red blood cells or a low level of functional hemoglobin. One of the many causes of anemia is destruction of red blood cells, a process called hemolysis (hemo means blood and lysis means disintegration). Any anemia caused by a shortened red blood cell survival in the circulation is called a hemolytic anemia. The causes of hemolytic anemia include septic shock, mechanical trauma, intrinsic red blood cell membrane defects or biochemical (enzyme) defects that make the cells more fragile, and antibody induced destruction. The antiglobulin test identifies red blood cells coated (sensitized) by antibody, and therefore, hemolytic anemia caused by immune mediated destruction. The test also identifies red blood cells coated by complement. Complement is a series of proteins that attach to the cell following the reaction of some types of antibodies with antigens on the cell surface. Complement components undergo changes after attachment to the red cells that damage the membrane and serve as binding sites for phagocytes that remove the red cells from the blood as they pass through the spleen and liver.
Immune-mediated hemolytic anemia can result from a transfusion reaction in which antibodies formed by the recipient attach to and destroy the donor's red blood cells, or it can result from the production of antibodies that attach to the surface of the person's own red cells (autoimmune hemolytic anemia).
Autoimmunity is the cause of many systemic collagen-vascular diseases, including rheumatoid arthritis and systemic lupus erythematosus, and several organ-specific diseases such as type I diabetes mellitus and chronic lymphocytic thyroiditis (hypothyroidism). Some persons with systemic autoimmune disease produce autoantibodies to their red cells, and the antiglobulin test is used to identify these antibodies. In addition, the antiglobulin test may be employed to detect the antibodies responsible for the destruction of the target tissue in autoimmune diseases.
Causes of immune-mediated hemolytic anemia include:
- drugs such as penicillin, methyldopa (lowers blood pressure ), and quinidine (treats heart rhythm disturbances)
- cancers of the lymph system, including Hodgkin's disease and lymphomas
- some viral infections
- collagen-vascular diseases
- incompatible blood transfusions
- Rh incompatibility between a mother and fetus (hemolytic disease of the newborn)
In some cases, the cause of an autoimmune hemolytic anemia cannot be identified.
A blood sample collected by venipuncture is used for antiglobulin tests. The nurse or phlebotomist collecting the specimen should observe universal precautions for the prevention of transmission of bloodborne pathogens. It is recommended that samples for the direct antiglobulin test be collected in EDTA. This chelates calcium, preventing the attachment of complement components to red cells during storage. Refrigeration of blood specimens that have cold agglutinins may cause a false positive test. The blood should be stored at room temperature until separation of red cells and serum or plasma.
There are two forms of the antiglobulin (Coombs') test. A direct antiglobulin (Coombs') test (DAT) detects antibody bound in vivo to either antigen(s) or complement components on the red cell surface. The test uses a broad spectrum reagent containing antibodies that bind to human immunoglobulin and C3. Such antibodies can be made by immunizing rabbits, burros, or goats with purified human immunoglbulin or complement proteins or by gene fusion technology (hybridomas) that produces a malignant clone of antibody secreting cells containing the genes needed to make the desired antibody. Reagents typically have reactivity to human IgG (the most common class of antibody), complement components C3b and C3d, and immunoglobulin light chains to permit detection of other immunoglobulin classes (IgA and IgM). A 2-3% suspension of the cells is made using saline. The blood cells are washed three times with isotonic saline to remove the immunoglobulins and other proteins not bound to the red cells. After washing the antiglobulin (AHG) reagent is added to the cell button. The cells are suspended in the reagent and the mixture is centrifuged. The cells are resuspended and examined for visible clumping (agglutination). If the red blood cells are coated with either antibody or complement they will clump, and the button will break up into small pieces instead of resuspending in the saline. If no agglutination is observed, a drop of antibody-coated red cells is added to the mixture and it is centrifuged again and examined. The presence of agglutination verifies that the antiglobulin reagent is present and had not been neutralized by improper washing. In addition, monospecific anti-human immunoglobulins may be used to distinguish between antibody and complement binding and to identify the class of antibody that has attached to the red blood cells. The direct antiglobulin tests can be used to detect autoantibodies that have attached to other types of cells. For example, the DAT test is often used to identify the autoimmune form of a kidney disease called glomerulonephritis. The antihuman immunoglobulin is conjugated to a fluorescent dye. This is added to a thin-section of the renal tissue on a microscope slide. The slide is washed to remove unbound antibody and then examined under a fluorescent microscope. Fluorescence of the tissue indicates a positive test for antibody-coated cells.
The second type of antiglobulin test is called the indirect antiglobulin test. This procedure is used to demonstrate the presence of specific antibodies in the serum or plasma. It is also used to detect blood group antigens of several blood group systems including the Kell, Kidd, and Duffy systems, to detect incomplete (nonagglutinating) antibodies that have attached to donor red blood cells in the compatibility test (cross-match), and to investigate the cause of a transfusion reaction. Many antibodies are able to attach to the corresponding antigens on red blood cells, but do not cause agglutination because the size of the antibodies is too small to cross link antigens on adjacent cells. These antibodies can be detected in plasma or serum by the indirect antiglobulin test. This test is performed in the same way is the direct test except that the red blood cells must be incubated with the patient's serum or plasma before washing and adding the AHG reagent. For example, in order to screen for antibodies that might be present in the serum of a person needing a blood transfusion, the serum is incubated with reagent red blood cells. Two or three different reagents are used each consisting of a standardized suspension of group O red cells obtained from various donors so that a broad spectrum of different blood group antigens is represented. Group O red cells are used because they lack A or B antigens that would cause agglutination with anti-A or anti-B antibodies that occur naturally in persons lacking the respective antigen. Following incubation, the red cells are washed to remove unbound proteins (antibodies) and the AHG is added. The cell suspension is centrifuged and examined for agglutination as described above. The presence of agglutination indicates that at least one antibody against the reagent cells are present. The indirect antiglobulin test using a fluorescent-labeled antihuman globulin is also used to identify antibodies produced against DNA and other cellular components by persons with various autoimmune diseases.
No preparation is needed for this test. Prior to performing the venipuncture, the nurse or other health care professional should document any medications the patient is currently taking, since many medications have been implicated in autoimmune hemolytic anemia.
The patient may feel discomfort when blood is drawn from a vein. Bruising may occur at the puncture site or the person may feel dizzy or faint. Pressure should be applied to the puncture site until the bleeding stops to reduce bruising. Warm packs can also be placed over the puncture site to relieve discomfort.
The most common complication is a bruise at the site of the puncture or excessive bleeding. The patient can apply moist warm compresses if there is any discomfort.
Antiglobulin tests are reported as negative or positive. If the direct antiglobulin test is positive, an elution study may be performed to identify the specificity of the antibody. In addition, the indirect antiglobulin test may be performed to determine if there is unattached circulating antibody. If the indirect antiglobulin test is positive, serial dilutions may be performed to quantify the concentration of antibody. The antibody titre is defined as the highest dilution of the serum that gives a positive test result. If the antiglobulin tests are negative, the anemia is unlikely to be immunemediated, and the physician will have to search elsewhere for a cause.
Health care team roles
A physician will order the tests, and will interpret the results. The blood specimen is drawn by a nurse or phlebotomist, and transported to the laboratory. AHG tests are performed by clinical laboratory scientists/medical technologists. If a direct AHG test is performed on tissue, the specimen is processed by a histologic technician and the tissue is examined under the microscope by a pathologist. The pathologist writes an interpretative report of the microscopic finding.
Anemia— Reduced oxygen-carrying capacity of the blood, due to too little hemoglobin or too few red blood cells.
Antibody— A protein made by the immune system, that is directed against antigens, and used as a weapon against foreign invaders in the body.
Antigen— The chemical that stimulates an immune response.
Collagen-vascular disease— Various diseases inflaming and destroying connective tissue.
Hematologist— Physician who specializes in diseases of the blood.
Hemoglobin— The red pigment in blood that carries oxygen.
Hemolysis— Breaking apart red blood cells.
Rh— A blood typing group, like the ABO system. When a mother is Rh negative and her baby is Rh positive, she may develop antibodies to the baby's blood that will cause it to hemolyze.
American Association of Blood Banks. Technical Manual, 13th ed., Bethesda, MD: American Association of Blood Banks, 1999.
American Society of Clinical Pathologists. Practical Diagnosis of Hematologic Disorders, 3rd ed., edited by Carl Kjeldsberg, et al. Chicago, IL: ASCP Press, 2000.
Henry, John B. Clinical Diagnosis and Management by Laboratory Methods, 20th ed. Philadelphia, PA: W. B. Saunders, 2001.
Rosse, Wendell and H. Franklin Bunn. "Hemolytic Anemias and Acute Blood Loss." In Harrison's Principles of Internal Medicine, edited by Kurt Isselbacher, et al. New York: McGraw-Hill, 1998.