The anti-human globulin test (coomb's) is divided into a direct test (direct reaction) and an indirect test (indirect reaction). The purpose of the direct test is to check for incomplete antibodies on the surface of red blood cells. Red blood cells with the corresponding antigen on their surface are called sensitized red blood cells after binding with incomplete antibodies. Sensitized erythrocytes washed with saline do not agglutinate in saline medium, but agglutination occurs when anti-human globulin serum is added, which is a positive direct test for anti-human globulin. This experiment is from Mudanjiang Medical College, undergraduate 5-year laboratory guide for testing majors.
Operation method
Anti-human globulin (coomb's) direct method test
Principle
The anti-human globulin test (coomb's) is divided into a direct test (direct reaction) and an indirect test (indirect reaction). The purpose of the direct test is to check for incomplete antibodies on the surface of red blood cells. Red blood cells with the corresponding antigen on their surface are called sensitized red blood cells after binding with incomplete antibodies. Sensitized erythrocytes washed with saline do not agglutinate in saline medium, but agglutination occurs when anti-human globulin serum is added, which is a positive direct test for anti-human globulin. Move Method I: Caveat 1. After the preparation of anti-human globulin serum, it should be identified by positive red blood cells or sensitized Rh-positive ¡°O¡± red blood cells, and can be used only if it is positive. For this reason, it is not necessary to make positive and negative controls for each test. 2. When the test is performed, the red blood cells are found to agglutinate before the unknown coomb's serum, which is mostly a phenomenon of cold agglutination, and the test should be performed at 37℃ to exclude the existence of cold antibodies. 3. When washing red blood cells, it is necessary to wash them three times with a large amount of saline in order to wash the serum proteins sufficiently to exclude the factors that inhibit a positive result. 4. There are two types of autoimmune antibodies, one is warm antibody, which is IgG reacting at room temperature and 37℃. One is warm antibody, which is IgG reacting at room temperature and 37℃, and the other is cold antibody, which is IgM reacting at 0-4℃. Therefore, it is necessary to perform the test according to the temperature environment. For more product details, please visit Aladdin Scientific website.
1. Examined red blood cells: take the patient's whole blood de-fibrinized (do not use anticoagulated blood, because anticoagulated blood can make the positive negative), and then wash the red blood cells with saline 3 times, and prepare 10% red blood cell suspension.
2. Dilute anti-human globulin serum (coomb's serum) into 1:2, 1:4, 1:8, 1:16 ...... and other multiples of the serum with saline, and add one drop of each serum into the concave slide.
3. Add an equal amount of 10% erythrocyte suspension from the patient and mix well. Place at room temperature for 15 minutes and observe whether there is agglutination or not, and report the positive result with the highest serum dilution.
Method II:
1. Take 3 test tubes and operate according to the table below:
2. Place 2. 3 tubes at 37°C for 1 hour, then wash all 3 tubes three times with large amounts of saline to form a 10% erythrocyte suspension.
3. Take four small test tubes and operate according to the table: 
4. After mixing. After mixing, set at room temperature for 3O minutes, observe the results, or low-speed centrifugation and observation.
5. results:
If the positive control agglutination, saline control and negative control are not agglutination means that the operation and reagents are not a problem, and then observation of the test tube report.