Direct Coombs Test Indirect Coombs Test

In this direct coombs test indirect coombs test post we have briefly explained about coombs test direct method and coombs test indirect method, principle, procedure, result, applications, and limitations.

The Coombs’ test is used to detect antibodies that act against the surface of your red blood cells. The presence of these antibodies indicates a condition known as hemolytic anemia, in which your blood does not contain enough red blood cells because they are destroyed prematurely. A healthy red blood cell lives for about 120 days; in people with hemolytic anemia, red blood cells are destroyed long before the 120-day marker. Sometimes, bone marrow can compensate for the early destruction of red blood cells by working overtime to make more of them. However, this extra effort may not be enough to combat hemolytic anemia.

coombs test direct and indirect

Direct Coombs Test Indirect Coombs Test

Hemolytic anemia can either be inherited genetically or acquired. An acquired form, autoimmune hemolytic anemia, is present in about 10% of people with lupus and results from an immune system attack on your red blood cells. People with AIHA may experience weakness, dizziness, fevers, and a yellowed complexion. Treatment calls for high doses of steroids such as prednisone, which may be supplemented with immunosuppressive medications such as azathioprine.

Principle

Red cells coated with complement or IgG antibodies do not agglutinate directly when centrifuged.  These cells are said to be sensitized with IgG or complement. In order for agglutination to occur an additional antibody, which reacts with the Fc portion of the IgG antibody, or with the C3b or C3d component of complement, must be added to the system. This will form a “bridge” between the antibodies or complement coating the red cells, causing agglutination.

Direct Coombs Test Indirect Coombs Test

1. Coombs Test Direct Method

Coombs test direct method is designed to identify maternal anti-Rh antibodies that are already bound to infant RBCs or antibodies bound to RBCs in patients with autoimmune hemolytic anemia.

Coombs Test Direct and Indirect

Coombs Test Direct Method

Procedure

1. Prepare a 5 % suspension in isotonic saline of the red blood cells to be tested. With clean pipette add one drop of the prepared cell suspension to a small tube.

2. Wash three times with normal saline to remove all the traces of serum. Decant completely after the last washing.

3. Add two drops of Anti-human serum. Mix well and centrifuge for one minute at 1500 RPM.

4. Resuspend the cells by gentle agitation and examine macroscopically and microscopically for agglutination.

2. Coombs Test Indirect Method

Coombs test indirect method is designed to identify Rh-negative mothers who are producing anti-Rh antibodies of the IgG isotype, which may be transferred across the placenta harming Rh-positive fetuses. Te indirect Coombs is also used in the diagnosis of transfusion reactions.

Coombs Test Direct and Indirect

Coombs Test Indirect Method

Procedure

1. Label three test tubes as T (test serum) PC (Positive control) and NC (negative control). In the tube labeled as T (Test), take 2 drops of test serum.

2. In the test tube labeled as PC (Positive control), take 1 drop of anti D serum. In the test tube labeled as NC (Negative control), take 1 drop of normal saline.

3. Add one drop of 5 % saline suspension of the pooled ‘O’ Rho (D) positive cells in each tube. Incubate all the three tubes for one hour at 37°C.

4. Wash the cells three times in normal saline to remove excess serum with no free antibodies, (in the case of inadequate washings of the red cells, negative results may be obtained).

5. Add two drops of Coombs serum (anti human serum) to each tube. Keep for 5 minutes and then centrifuge at 1,500 RPM for one minute. Resuspend the cells and examine macroscopically as well as microscopically.

Results Interpretation

Coombs Test Negative

No clumping of cells (no agglutination). This means you have no antibodies to red blood cells.

Coombs Test Positive

Clumping (agglutination) of the blood cells during a coombs test direct method means that you have antibodies on the red blood cells and that you may have a condition that causes the destruction of red blood cells by your immune system (hemolysis). This may be due to

Hemolytic anemia,

Chronic lymphocytic leukemia or similar disorder,

Erythroblastosis fetalis (hemolytic disease of the newborn),

Infectious mononucleosis,

Mycoplasmal infection,

Syphilis,

Systemic lupus erythematosus and

Transfusion reaction, such as one due to improperly matched units of blood.

Application

Coombs Test Direct Method

1. Coombs test direct method  is used primarily to help determine whether the cause of hemolytic anemia is due to antibodies attached to RBCs. Hemolytic anemia is a condition in which red blood cells (RBCs) are destroyed more quickly than they can be replaced.

2. Coombs test direct method may also be used to help diagnose hemolytic disease of the newborn (HDN) due to an incompatibility between the blood types of a mother and baby. Coombs test direct method may also be used to investigate a suspected transfusion reaction.

Coombs Test Indirect Method

1. Coombs test indirect method is administered to determine if there was a potential bad reaction to a blood transfusion. The indirect Coombs test is used in prenatal testing of pregnant women.

2. Blood banks use the coombs test indirect method to determine whether there is likely to be an adverse reaction to blood to be transfused.

Limitations

1. Sometimes, especially in older adults, a Coomb’s test will have an abnormal result even without any other disease or risk factors, and test can only be rarely used to diagnose a medical condition.

Further Readings