Direct Antiglobulin Testing

The direct antiglobulin test (DAT) is used to determine whether red blood cells (RBCs) have been coated in vivo with immunoglobulin, complement, or both. The direct antiglobulin test is sometimes colloquially referred to as the direct Coombs test, because it is based on a test developed by Coombs, Mourant, and Race. ^[1]

By way of comparison, the indirect antiglobulin test (IAT), colloquially referred to as the indirect Coombs test, is used to determine the presence of antibody in the serum or plasma. ^[2]

There are many causes of a positive direct antiglobulin test.

Depending on the technique and the reagents used, a positive direct antiglobulin test has been reported in 1:1000 to 1:14,000 blood donors and 1%-15% of hospital patients. Most blood donors with positive direct antiglobulin test results appear healthy, and most show no overt signs of hemolytic anemia. ^[3]

It is important to remember that a positive direct antiglobulin test is neither 100% sensitive nor specific for hemolytic anemia.

The clinical significance of a direct antiglobulin test result should take into consideration the patient's clinical history, diagnoses, and other laboratory test results.

The direct antiglobulin test is used most commonly to investigate possible hemolytic transfusion reactions, hemolytic disease of the fetus and newborn (HDFN), autoimmune hemolytic anemia, and drug-induced immune hemolysis. ^{[4, 5]}

Hemolytic transfusion reactions

A positive direct antiglobulin test result may be the first indication of an immune response to a recent transfusion. ^[4]

The patient's developing antibodies will coat transfused RBCs bearing the corresponding antigen, resulting in a positive direct antiglobulin test result.

Antibodies appear within 7-10 days after a primary exposure or within 1-2 days of a secondary response.

Antibodies can shorten the survival of previously or subsequently transfused RBCs.

Hemolytic disease of the fetus and newborn

Hemolytic disease of the fetus and newborn is the destruction of fetal and newborn RBCs by maternal immunoglobulin G (IgG) alloantibodies that cross via the placenta into the fetal circulation. ^[4]

Maternal alloantibodies are specific for inherited paternal RBC antigens expressed by fetal RBCs.

Maternal alloantibodies bind to the corresponding antigen on fetal RBCs, resulting in a positive direct antiglobulin test result.

Autoimmune hemolytic anemia

Conditions in this category include warm autoimmune hemolytic anemia, cold agglutinin syndrome, mixed- or combined-type autoimmune hemolytic anemia, and paroxysmal cold hemoglobinuria. ^[4]

Warm autoimmune hemolytic anemia is the most common type of autoimmune hemolytic anemia and is generally associated with warm-reactive IgG autoantibodies. Three patterns of reactivity may be found on a direct antiglobulin test: IgG alone, complement alone, or both.

Cold agglutinin syndrome is associated with cold-reactive autoantibodies, most frequently immunoglobulin M (IgM). In areas of low temperature in the peripheral circulation, IgM binds to the RBCs and causes complement to also adhere to the RBCs. As the RBCs circulate to areas of higher temperature, the IgM may dissociate, but the complement remains. Additionally, the standard direct antiglobulin test will not detect IgM coating of RBCs. As such, complement alone is commonly detected on the direct antiglobulin test.

In a patient with mixed-type autoimmune hemolytic anemia, the direct antiglobulin test commonly detects both IgG and C3.

Paroxysmal cold hemoglobinuria is caused by a biphasic hemolysin, known as the Donath-Landsteiner hemolysin. This autoantibody is an IgG complement-fixing antibody that reacts with RBCs in the colder areas of the circulation. The IgG causes C3 to bind irreversibly to RBCs; subsequently, the IgG dissociates from RBCs in warmer parts of the body. Thus, the direct antiglobulin test is typically positive only for complement.

Drug-induced hemolysis

Drugs may cause a positive direct antiglobulin test result and/or immune-mediated hemolysis, with an incidence of approximately 1 in 1 million. ^[4]

Drugs can induce the formation of antibodies, either against the drug itself or against intrinsic RBC antigens or the RBC membrane. This may result in a positive direct antiglobulin test result, immune hemolysis, or both.

Drugs commonly implicated in this include penicillin, second- and third-generation cephalosporins, alpha-methyldopa, and procainamide, among others.

The direct antiglobulin test (DAT) is valuable in the diagnosis of the following: ^[4]

• Autoimmune hemolytic anemia

• Drug-induced immune hemolytic anemia

• Hemolytic transfusion reactions due to alloantibodies

• Hemolytic disease of the fetus and newborn

• Systemic lupus erythematosus (in the absence of hemolytic anemia)

RBC autoantibodies and alloantibodies are usually IgM or IgG antibodies, occasionally IgA or mixture of each immunoglobulin. Pentameric IgM causes direct agglutination in saline-suspended RBCs. By contrast, IgG antibodies adhere to the corresponding antigens on the RBC membrane, but they do not result in agglutination. RBCs with adherent IgG antibodies can be considered "sensitized."

Based on work by Coombs and others, it was postulated that, if rabbits were injected with human gamma globulin, they would develop antibodies to the foreign protein, so-called antihuman globulin (AHG). After suitable processing, this AHG would react specifically with IgG adhering to RBC membranes, cross-linking the sensitized RBCs and causing agglutination.

Both polyspecific and monospecific AHG is available commercially. Polyspecific AHG contains anti-IgG and anti-C3d. Monospecific AHG contains either a monospecific anti-IgG or an anti-C3 containing anti-C3d activity. Positive direct antiglobulin test results with a polyspecific AHG should be tested further with monospecific reagents. This additional testing provides further information in determining the type of immune hemolytic anemia involved. ^[3]

Positive direct antiglobulin test result

The following indicate a positive direct antiglobulin test result: ^[4]

• Agglutination observed after immediate centrifugation (usually seen when patient RBCs are coated with IgG)

• Agglutination observed after centrifugation following room-temperature incubation (usually seen when patient RBCs are coated with complement)

There are many causes of a positive direct antiglobulin test, including the following: ^{[3, 4]}

• Autoantibodies to intrinsic RBC antigens

• Hemolytic transfusion reactions

• Hemolytic disease of the fetus and newborn

• Drug-induced antibodies

• Passively acquired alloantibodies (eg, from donor plasma, derivatives, or immunoglobulin)

• Nonspecifically adsorbed proteins (eg, hypergammaglobulinemia, high-dose intravenous immunoglobulin [IVIG], modification of RBC membranes by drugs)

• Complement activation due to bacterial infection, autoantibodies, or alloantibodies

• Antibodies produced by passenger lymphocytes (eg, in transplanted organs or hematopoietic components)

In the case of a positive direct antiglobulin test, three additional tests may be helpful. ^[4]

Monospecific AHG reagents can confirm which globulins are present.

The serum/plasma can be tested to detect and identify clinically significant antibodies to red cell antigens.

An eluate can be prepared from the sensitized red cells. An eluate displaces antibody from sensitized RBCs and recovers antibody in a usable form. The displaced antibody is then tested against a panel of reagent RBCs to determine the activity of the antibody. Eluate preparation frequently concentrates small amounts of antibody and may facilitate identification of weakly reactive antibody. When the eluate reacts with all the reagent RBCs tested, the patient most likely has an autoantibody. This is particularly true if the patient has not been recently transfused.

Negative direct antiglobulin test result

The result is negative if no agglutination is observed at either test phase, but agglutination is observed after known IgG-coated and complement-coated RBCs are added to the appropriate tubes. ^[4]

In the case of a negative direct antiglobulin test, the known IgG-coated RBCs act as a positive control. If the known IgG-coated RBCs do not agglutinate, the test finding is presumed to be a false-negative result and must be repeated. ^[6]

Polyspecific and anti-IgG reagents are very sensitive and can detect as few as 200-500 molecules of IgG per RBC. However, patients may experience autoimmune hemolytic anemia when IgG coating is below this level. As such, the direct antiglobulin test (DAT) result be negative despite the presence of IgG-coated RBCs. ^[5] This entity is sometimes referred to as direct antiglobulin test–negative autoimmune hemolytic anemia. ^[7] Therefore, even if the result is negative, elution may be performed if immune hemolysis is suspected.

If all of the test tubes and the negative control demonstrate agglutination, spontaneous agglutination is likely occurring. This may be caused by IgM coating. Spontaneous agglutination must be resolved before further testing. ^{[4, 8]}

False-positive results may be caused by the following:

• Overcentrifugation or contaminated reagents ^[8]

• Insufficient washing of the patient's RBCs ^[8]

• If the test tubes were left to stand following centrifugation or if the RBCs were left in suspension for an extended period before testing ^[8]

Hemolytic transfusion reactions may result in a positive direct antiglobulin test result if sensitized RBCs have not been destroyed or a negative result if hemolysis and rapid clearance have occurred. ^[4]

The direct antiglobulin test is performed by tube agglutination, as follows: ^{[4, 8]}

1. Patient RBCs are acquired from an ethylenediaminetetraacetic acid (EDTA)–anticoagulated blood sample

2. One drop of a 2%-5% suspension of patient RBCs (in saline or native plasma) is dispensed into each of 4 test tubes

3. The patient RBCs are washed 3-4 times with saline, and the final wash supernate is completely decanted

4. Polyspecific AHG reagent is added to 1 tube

5. Similarly, tests with the anti-IgG and anti-C3 reagents are set up

6. Two drops of saline are added to the fourth tube

7. The contents of each tube are gently agitated

8. Centrifuge (Note: For anti-complement, some manufacturers may recommend a delay before centrifugation; others may recommend that initial negative results with anti-C3 be re-evaluated following incubation at room temperature for 5 minutes and then recentrifugation)

9. The RBCs are examined for agglutination; the results are graded and recorded

10. IgG-coated RBCs are added to all negative tests with anti-IgG or polyspecific AHG; complement-coated RBCs are added to all negative tests with anti-C3; recentrifuge and examine the tests macroscopically for agglutination

11. All tests are repeated when IgG-coated RBCs and/or complement-coated RBCs are nonreactive