What is the procedure for performing a direct Coombs test (Direct Antiglobulin Test)?

From the Guidelines

The Direct Coombs test, also known as the Direct Antiglobulin Test (DAT), should be performed by collecting a blood sample, washing the red blood cells, and then adding anti-human globulin reagent, with the most critical step being the proper preparation of the sample to ensure accurate results and minimize the risk of morbidity and mortality due to incorrect diagnoses or delayed treatment. To perform the test, first collect 2-5 mL of blood in an EDTA tube, as this is a standard procedure for preventing clotting and preserving the sample for testing 1. The blood sample should be processed as quickly as possible, ideally within 1 hour of collection, to prevent loss of coagulation factors and ensure the accuracy of the test results 1. The sample should undergo double centrifugation to ensure that it is platelet-poor, which can be achieved by centrifuging the sample at 2500 ×g for 15 minutes at room temperature, collecting the supernatant, and then recentrifuging it at the same speed for an additional 10 minutes 1. After the final centrifugation, the plasma should be frozen at -80 °C immediately to preserve the coagulation proteins, which are stable for a minimum of 2 years at this temperature 1. Before analysis, the frozen plasma should be thawed at 37 °C to avoid the formation of cryoprecipitate, and then mixed thoroughly before testing 1. The test itself involves washing the red blood cells three times with saline solution to remove any free antibodies, and then adding anti-human globulin reagent to detect any antibodies or complement bound to the red blood cells 1. The results of the test should be interpreted with caution, taking into account the potential for false positives or false negatives, and should be confirmed with additional testing if necessary to ensure accurate diagnosis and treatment. Some key points to consider when performing the Direct Coombs test include:

• The importance of proper sample preparation to ensure accurate results
• The need for double centrifugation to remove platelets and prevent interference with the test
• The importance of freezing the plasma at -80 °C to preserve coagulation proteins
• The need for careful interpretation of the test results to ensure accurate diagnosis and treatment. By following these steps and considering these key points, the Direct Coombs test can be a valuable tool for diagnosing and managing conditions such as autoimmune hemolytic anemia, hemolytic disease of the newborn, and transfusion reactions, and can help to improve patient outcomes and reduce morbidity and mortality.

From the Research

Procedure for Performing a Direct Coombs Test

The direct Coombs test, also known as the direct antiglobulin test (DAT), is a laboratory procedure used to detect the presence of immunoglobulin or complement on the surface of red blood cells [ 2, 3 ].

Steps Involved in the Test

• The test involves the use of antihuman globulin (AHG) reagent, which can be either polyspecific or monospecific [ 4 ].
• The AHG reagent is used to detect the presence of immunoglobulin or complement on the surface of red blood cells.
• The test can be performed using a gel card system, which has made the procedure and interpretation of the DAT simpler [ 4 ].
• The gel card system uses a polyspecific AHG reagent to detect the presence of immunoglobulin or complement on the surface of red blood cells.
• The test can also be performed using a manual tube method, which uses a monospecific AHG reagent to detect the presence of immunoglobulin or complement on the surface of red blood cells [ 4 ].

Interpretation of Results

• A positive direct Coombs test indicates the presence of immunoglobulin or complement on the surface of red blood cells [ 2, 3 ].
• The test results can be interpreted as positive or negative, and the intensity of the reaction can be graded [ 5 ].
• False-positive and false-negative results can occur, and the test results should be correlated with clinical presentation and other laboratory findings [ 4, 6 ].

Special Considerations

• In cases where the direct Coombs test is negative, but clinical presentation suggests autoimmune hemolytic anemia, alternative methods can be used to detect low levels of IgG sensitization [ 6 ].
• Eluate testing can be performed following a microscopically positive direct Coombs test to identify new antibodies [ 5 ].