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Coombs Test Guide for Lab Technicians

The Coombs test, also known as the direct antiglobulin test, is performed to detect antibodies attached to red blood cells. There are two types of Coombs tests: the indirect Coombs test detects antibodies in a patient's serum, while the direct Coombs test detects antibodies attached to the surface of the patient's red blood cells. Both tests involve washing red blood cells, adding serum to look for agglutination, which would indicate the presence of antibodies. The Coombs test is used to detect Rh antibodies in pregnant women, antibodies in newborn babies, and to diagnose other conditions where red blood cells are sensitized by drugs, transfusions, or autoimmune disorders.

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Freddy Vallejo Leon
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588 views11 pages

Coombs Test Guide for Lab Technicians

The Coombs test, also known as the direct antiglobulin test, is performed to detect antibodies attached to red blood cells. There are two types of Coombs tests: the indirect Coombs test detects antibodies in a patient's serum, while the direct Coombs test detects antibodies attached to the surface of the patient's red blood cells. Both tests involve washing red blood cells, adding serum to look for agglutination, which would indicate the presence of antibodies. The Coombs test is used to detect Rh antibodies in pregnant women, antibodies in newborn babies, and to diagnose other conditions where red blood cells are sensitized by drugs, transfusions, or autoimmune disorders.

Uploaded by

Freddy Vallejo Leon
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPT, PDF, TXT or read online on Scribd

COOMBS TEST

Indirect Coombs test (Indirect Antiglobulin test):


This test is performed to detect presence of Rh-antibodies or other antibodies in patients serum in case of the following: 1. To check whether an Rh-negative women (married to Rhpositive husband) has developed Anti Rh-antibodies

2. Anti D may be produced in the blood of any Rh-negative person by exposure to D antigen by

Transfusion of Rh positive blood Pregnancy, if infant is Rh positive (if father is Rh-positive) Abortion of Rh-positive fetus.

Requirements:

Test tubes: (10x75 mm) Pasteur pipettes Incubator Centrifuge

Specimen: Serum (need not be fasting) Reagents: 1. Antihuman serum 2. Anti-D serum Additional Requirements: Coombs control cells
A. Make a pooled O Rho (D) positive cells from at least three different O positive blood samples. B. Wash these cells three times in normal saline (these cells should be completely free from serum with no free antibodies). Make 5 % saline suspension of these cells

Procedure:
1.

Label three test tubes as T (test serum) PC (Positive control) and NC (negative control). In the tube labelled as T, add two drops of Anti-D serum

2.

3.
4.

In the tube PC add one drop of saline


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 37C

5.

Procedure: (cont.)

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). 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

Test Interpretation:
Observations 1 Positive Control (PC)
(A)

Conclusions Correctly performed test procedure. Coombs serum may not be proper. Repeat the test again.

Agglutination No Agglutination

(B)

Negative control It should show no agglutination, since saline does not contain Anti-D or any other antibodies. (NC) Test (Serum) (T)
(A)

Agglutination (and if PC results are correct) No Agglutination

Patients serum contains Anti-D.

(B)

Patients serum does not contain Anti-D.

Direct Coombs test (direct antiglobulin test):

This test is performed to detect anti-D antibody or other antibodies attached to the red cell surface within the blood stream. This occurs in the following circumstances:

When there is a Rh-positive baby in the womb of a sensitized Rhnegative women; the antibodies produced in the mothers serum cross the placenta and after entering the baby's blood stream, these antibodies will attach to the baby's Rh-positive red blood cells. These coated (or sensitized) cells are clumped and removed from the circulation, causing hemolytic anemia (Hemolytic Disease of the Newborn: Erythroblastosis Fetalis). When the baby is born, the baby's blood is collected (or cord blood is collected from umbilical cord) and tested by the anti globulin Coombs test (direct) to detect anti D antibodies coated on red blood cells. Transfusion reactions Drug induced red cells sensitization Autoimmune hemolytic anemia

Requirements: (same as that for Indirect Coombs test)


Test tubes: (10x75 mm) Pasteur pipettes Incubator Centrifuge

Specimen: Blood drawn into EDTA is preferred but oxalateed, or clotted, citrated whole blood may be used (specimen need not be fasting sample)

Procedure:
1.

Prepare a 5 % suspension in isotonic saline of the red blood cells to be tested.

2.

With clean Pasture pipette add one drop of the prepared cell suspension to a small tube.
Wash three times with normal saline to remove all the traces of serum.

3.

4.
5. 6. 7.

Decant completely after the last washing


Add two drops of Antihuman serum. Mix well and centrifuge for one minute at 1500 RPM. Resuspend the cells by gentle agitation and examine macroscopically and microscopically for agglutination.

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