Lecture #6: THE ANTIGLOBULIN (COOMBS) REACTION

The antiglobulin (Coombs test) is used primarily to detect globulins immunologically bound to red cells. Antiglobulin reaction can be shown through the use of radioactive labeled anti–Rh_o (D) that only 300 molecules of IgG per red cell maybe sufficient to produce agglutination in the antiglobulin reaction. A recent modification of the antiglobulin reaction, the complement fixing antibody consumption test (CFAC), which currently is a research procedure, is capable of detecting fewer than 35 molecules of IgG per cell. Red cells from patients with acquired immune hemolytic anemia with a negative antiglobulin reaction by conventional method, when tested with CFAC where shown to have abnormal amounts of cell bound IgG (from 70 to 434 molecules per cell). Red cells coated with fewer than 35 molecules of IgG per cell where considered to represent non–immunologically bound IgG. It appears that the conventional antiglobulin reaction, despite its inherent sensitivity, has blind spot and may fail to detect immunologically significant red cell sensitization when the red cells are coated with 35 to 300 molecules of IgG.

Types of Anti–Human Globulin Reagent

1. Polyspecific or multivalent or broad spectrum reagent. This is the basic type of reagent used in routine blood banking procedures, such as compatibility testing and antibody screening. It contains anti–IgG and anti–C3d (anti–complement component). The reagent may contain antibodies of other specificities, such as anti–IgM, anti–IgA, anti–c3b or anti–C4. If the test is positive with polyspecific sera, monospecific sera may be used for follow up testing.

2. Monospecific or monovalent reagent. Example of this is the anti–IgG and the anticomplement C3b + C3d (previously called anti–non–gamma). Anti–IgG can detect most clinically significant antibodies. This type of reagent is useful in differentiating agglutination produced by IgG antibodies rather than agglutination due to complement fixation produced by cold agglutinins. Although various components of complement can sensitize erythrocytes, the C3d component is associated with immune hemolysis; therefore, it is important that this component be included in anti–complement reagents to facilitate investigation of immune hemolytic anemias. Monospecific sera containing anti– IgM or anti–IgA are not routinely used.

Reagent preparation

In the traditional preparation of anti–human globulin reagent, one colony of rabbits is injected with purified gamma globulin and a second colony is injected with purified component of human beta globulin. After a suitable time, plasma is harvested from the whole blood of the rabbits as the raw material. It is pooled and manufactured into a specific reagent for use in detecting human gamma globulin or component of complement. The product is buffered with bovine albumin and 0.1% sodium azide is added as preservative. If color is added such as ortho anti–human globulin (green), FD & C blue no. 1 and FD & C yellow no.5 dye are added.

Main application of the Antiglobulin Test

A. Direct Antiglobulin Test (DAT)

The DAT is used for the detection of in vivo red blood cell sensitization. Washed red blood cells from the patient are directly tested with antiglobulin serum.

The direct antiglobulin test is useful in:

1.  Diagnosis of hemolytic disease of the newborn

2.  Diagnosis of autoimmune hemolytic anemia

3. Diagnosis of red cell sensitization reaction caused by drugs (penicillin, cephalosporin, and alpha–methyl dopa)

4.  Investigation of transfusion reaction.

False positive DAT is seen:

1.  Extreme reticulocytosis

2.  Lead poisoning

3.  Drug–induced hemolysis

4.  Viral diseases

B. Indirect Antiglobulin Test

The indirect antiglobulin test is used for the detection of antibodies that may cause red blood cell sensitization in vitro. The antibody–containing serum is incubated with specified red blood cell which, following washing are reacted with antiglobulin serum to see whether red blood cell sensitization has occurred.

The indirect antiglobulin test is useful in:

1. Cross matching

2. Detection and identification of unexpected antibodies.

3. Detection of antigens not demonstrable by other technique.

4. Investigative studies, such as antiglobulin consumption test, mixed agglutination reaction and leukocyte or platelet studies.

Factors affecting the antiglobulin test

1. Sensitization phase (in vitro only)

a.  Temperature

b.  Medium (saline, albumin, serum or enzyme)

c.  Time of incubation

d.  Proportion of serum to cells

2. Washing phase – it should be rapid and uninterrupted to minimize loss of cell bound antibody by elution

3. Effects of centrifugation

4. Methods of reading results

5. Controls used

Sources of error

A. False negative results

1. Inadequate washing of red cells result in neutralization of the antiglobulin serum by trace amounts of residual globulin.

2. Contamination with human serum will neutralize the reagent.

3. Elution of antibody from the red cells may take place if the test is interrupted or delayed, particularly during the washing phase.

4. The optimum temperature for reactivity of the antibody must be maintained during incubation to achieve maximal coating of the cells.

5. A cell suspension that is too heavy will not permit optimum coating with the antibody; if too weak, reading agglutination may be difficult. A 2% to 5% suspension of RBC is preferred.

6. Test cells, test serum and antiglobulin serum lose reactivity if improperly stored.

7. Some antibodies may be detected only in the presence of active complement. Anticoagulants such as EDTA will chelate calcium, preventing activation of complement. Thus, the use of plasma rather than serum may lead to a false negative reaction. Old serum will also have impaired complement activity.

8. A prozone reaction should not be a problem with licensed products. Standardization is done by the manufacturer and his directions for the test must be followed.

9.  Antiglobulin serum may have been omitted.

10.  Undercentrifugation or overcentrifugation.

11.  Insufficient incubation time.

12.  Failure to check negative reactions, microscopically.

B. False positive results

1. Cells have a positive direct antiglobulin test cannot be used with reagent antiserum that require an antiglobulin phase, because all such cells will be agglutinated by the antiglobulin serum.

2.  Bacterial contamination of test cells or septicemia.

3. Extreme reticulocytosis because of transfusion bound to reticulocytes reacting with antitransferrin in the globulin reagent.

4. Saline stored in glass bottle may contain colloidal silica leached from the container.

5. Saline stored in metal containers or used in equipment with metal parts, may contain metallic ions which may bring about nonspecific protein–coating of the red cells.

6. Improperly prepared antiglobulin serum may contain traces of species specific antibodies.

7. When all the antiglobulin tests are weakly positive, the cause may be improperly cleaned glasswares or other forms of contamination.

8. Overcentrifugation.

9. Patient’s or donor’s serum contain a naturally occurring cold autoantibody that can sensitize their own or other complement

10. Red blood cells may be autoagglutinated before they are washed, and this agglutination may persist through washing leading to a false positive reaction when antiglobulin serum is added.