There are two methods of detecting and characterizing antibodies: (1) the direct Coombs’ test and (2) a group of procedures that try to determine if an antibody is present, and if present, attempt to identify the antibody by showing what the antibody will do in various controlled conditions.

Direct Coombs’ test

To prepare reagents for the Coombs’ test, human globulin, either gamma (IgG), nongamma (IgM), or a mixture of the two, is injected into rabbits. The rabbit produces antibodies against the injected human globulin. Rabbit serum containing these antihuman globulin antibodies is known as Coombs’ serum. Since human antibodies are globulin, usually gamma globulin, addition of Coombs’ serum (rabbit antibody against human gamma globulin) to anything containinghuman antibodies will result in the combination of the Coombs’ rabbit antibody with human antibody. There also has to be some indicator system that reveals that the reaction of the two antibodies has taken place. This can be seen visually if the Coombs’ rabbit antibody has been tagged with a fluorescent dye; or if the reaction takes place on the surface of RBCs, lysis or agglutination of the RBC can be produced.

The direct Coombs’ test demonstrates that in vivo coating of RBCs by antibody has occurred. It does not identify the antibody responsible. It is a one-stage procedure. The Coombs’ serum reagent is simply added to a preparation of RBCs after the RBCs are washed to remove nonspecific serum proteins. If the RBCs are coated with antibody, the Coombs’ reagent will attack this antibody on the RBC and will cause the RBCs to agglutinate to one another, forming clumps. The antibody on the RBC is most often univalent but sometimes is polyvalent. Although antibodies on RBCs that are detected by the direct Coombs’ test are most often antibodies to RBC blood group antigens, certain medications (e.g., methyldopa and levodopa) in some patients may cause autoantibodies to beproduced against certain RBC antigens. Also, in some cases antibodies not directed against RBC antigens can attach to RBCs, such as antibodies developed in some patients against certain medications such as penicillin or autoantibodies formed in the rheumatoid-collagen diseases or in some patients with extensive cancer. In addition, some reports indicate an increased incidence of apparently nonspecific positive direct Coombs’ reactions in patients with elevated serum gamma globulin levels.

The reagent for the direct Coombs’ test can be either polyspecific or monospecific. The polyspecific type detects not only gamma globulin but also the C3d subgroup of complement. Complement may be adsorbed onto RBCs in association with immune complexes generated in some patients with certain conditions, such as the rheumatoid-collagen diseases and certain medications, such as quinidine and phenacetin. Monospecific Coombs’ reagents are specific either for IgG immunoglobulin (and therefore, for antibody) or for complement C3d. If the polyspecific reagent produces a positive result, use of the monospecific reagents (plus elution techniques discussed later) can narrow down the possible etiologies.

The direct Coombs’ test may be done by either a test tube or a slide method. The direct Coombs’ test must be done on clotted blood and the indirect Coombs’ test on serum, since laboratory anticoagulants may interfere. A false positive direct Coombs’ test result may be given by increased peripheral blood reticulocytes using the test tube method, although the slide technique will remain negative. Therefore, one should know which method the laboratoryuses for the direct Coombs’ test.

In summary, positive direct Coombs’ test results can be due to blood group incompatibility, may be drug induced, may be seen after cardiac valve operations, and may appear in rheumatoid-collagen diseases, malignancy, idiopathic autoimmune hemolytic anemia, and other conditions. The overall incidence of a positive direct Coombs’ test result in hospitalized patients is reported to be about 7%-8% (range, 1%-15%).

The main indications for the direct Coombs’ test include the following (most are discussed later in detail):

1. The diagnosis of hemolytic disease of the newborn.
2. The diagnosis of hemolytic anemia in adults. These diseases include manyof the acquired autoimmune hemolytic anemias of both idiopathic and secondary varieties. Results of the direct Coombs’ test at normal temperatures are usually negative with cold agglutinins.
3. Investigation of hemolytic transfusion reactions.

In these clinical situations the indirect Coombs’ test should not be done if the direct test result is negative, since one is interested only in those antibodies that are coating the RBCs (and thus precipitating clinical disease).

Antibody detection and identification

Indirect Coombs’ test. The indirect Coombs’ test is a two-stage procedure. The first stage takes place in vitro and may be done in either of two ways:

1. RBCs of known antigenic makeup are exposed to serum containing unknown antibodies. If the antibody combines with the RBCs, as detected by the second stage, this proves that circulating antibody to one or more antigens on the RBC is present. Since the RBC antigens are known, this may help to identify that antibody more specifically.
2. Serum containing known specific antibody is exposed to RBCs of unknown antigenic makeup. If the antibody combines with the RBCs, as detected by the second stage, this identifies the antigen on the RBCs.

The second stage consists in adding Coombs’ serum to the RBCs after the RBCs have been washed to remove nonspecific unattached antibody or proteins. Ifspecific antibody has coated the RBCs, Coombs’ serum will attack this antibody and cause the cells to agglutinate. The second stage is thus essentially adirect Coombs’ test done on the products of the first stage.

Therefore, the indirect Coombs’ test can be used either to detect free antibody in a patient’s serum or to identify certain RBC antigens, depending on how the test is done.

The major indications for the indirect Coombs’ test are the following:

1. Detection of certain weak antigens in RBCs, such as Du or certain RBC antigens whose antibodies are of the incomplete type, such as Duffy or Kidd (see antibody screen).
2. Detection of incomplete antibodies in serum, either for pretransfusion screening or for purposes of titration.
3. Demonstration of cold agglutinin autoantibodies.

The indirect Coombs’ test is almost never needed routinely. In most situations, such as cold agglutinins or antibody identification, simply ordering atest for these substances will automatically cause an indirect Coombs’ test to be done. The indirect Coombs’ test should be thought of as a laboratory technique rather than as an actual laboratory test.

False positives and false negatives may occur with either the direct or indirect Coombs’ technique due to mixup of patient specimens, clerical error when recording results, technical error (too much or not enough RBC washing; also failure to add reagents or adding the wrong reagent), contamination by 5% or 10% glucose in water (but not glucose in saline) from intravenous tubing, and, rarely, use of faulty commercial Coombs’ reagent.

Antibody elution. When a direct Coombs’ test yields positive results, especially when thecause is thought to be a blood group–specific antibody, it is desirable to attempt elution (removal or detachment) of the antibody from the RBC to determine the antigen against which it is reacting. This is usually done by changing the physical conditions surrounding the antibody to neutralize the attachment forces. The most common current methods are heat, freeze-thaw, and chemical. Once the antibody is isolated from the RBCs, it can be tested with a panel of RBCs containing known antigens to establish its identity.